Oligonucleotide analogues and methods utilizing the same

ABSTRACT

A method for the prevention or treatment in a mammal of a disease preventable or treatable by the pharmacologically useful antisense or antigene activity of an oligonucleotide analogue or a pharmacologically acceptable salt thereof in the body of said mammal, which method comprises administering to said mammal in need of such prevention or treatment a pharmaceutically effective amount of an oligonucleotide analogue comprising two or more nucleoside units, wherein at least one of said nucleoside units is a structure of the formula (2): 
                         
wherein A is methylene; and B is an unsubstituted purin-9-yl, an unsubstituted 2-oxo-pyrimidin-1-yl or a substituted purin-9-yl; or a pharmacologically acceptable salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of application Ser. No.11/881,775 filed Jul. 27, 2007, now U.S. Pat. No. 7,816,333 which is acontinuation application of application Ser. No. 10/430,705 filed May 5,2003 (U.S. Pat. No. 7,314,923), which is a divisional application ofapplication Ser. No. 09/925,673 filed Aug. 9, 2001 (U.S. Pat. No.7,335,765), which is a continuation-in-part application of Internationalapplication PCT/JP00/00725 filed Feb. 10, 2000 (not published inEnglish), the entire contents of each of the aforesaid application Ser.No. 11/881,775, Ser. No. 10/430,705, Ser. No. 09/925,673 andPCT/JP00/00725 is hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel oligonucleotide analogues, whichexhibit antisense or antigene activity having excellent stability, orexhibit excellent activity as a detection agent (probe) for a specificgene or as a primer for starting amplification, and to novel nucleosideanalogues which are intermediates for their production.

2. Background Information

Oligonucleotide analogues, which have excellent antisense or antigeneactivity and which are stable in the body are expected to be usefulpharmaceuticals. In addition, oligonucleotide analogues having a highdegree of stable complementary chain formation ability with DNA or mRNAare useful as detection agents for a specific gene or as primers forstarting amplification.

In contrast, naturally-occurring oligonucleotides are known to bequickly decomposed by various nucleases present in the blood and cells.In some cases, naturally-occurring oligonucleotides may not havesufficient sensitivity for use as detection agents for specific genes oras primers for starting amplification due to limitations on theiraffinity with complementary base sequences.

In order to overcome these shortcomings, various non-naturally-occurringoligonucleotide analogues have been produced, and have been attempted tobe developed for use as pharmaceuticals or detection agents for specificgenes. Namely, known examples of such non-naturally-occurringoligonucleotide analogues include those in which an oxygen atom attachedto a phosphorus atom in a phosphodiester bond of an oligonucleotide isreplaced with a sulfur atom, those in which said oxygen atom is replacedwith a methyl group, those in which said oxygen atom is replaced with aboron atom, and those in which a sugar moiety or base moiety of anoligonucleotide is chemically modified. For example, ISIS Corp. hasdeveloped thioate-type oligonucleotide ISIS2922 (Vitravene) as atherapeutic agent for human cytomegalovirus retinitis and ISIS2922 hasbeen put on the open market in the United States.

However, in consideration of the potency of the antisense or antigeneactivity in the above non-naturally-occurring oligonucleotide analogues,namely the ability to form a stable complementary chain with DNA ormRNA, stability with respect to various nucleases, and the manifestationof adverse side effects due to non-specific bonding with variousproteins in the body, there has been a need for anon-naturally-occurring oligonucleotide analogue having even betterstability in the body, a low incidence of adverse side effects and ahigh ability to form complementary chains.

SUMMARY OF THE INVENTION

The inventors of the present invention conducted intensive research overa long period of time on non-naturally-occurring oligonucleotideanalogues having excellent antisense or antigene activity, excellentstability in the body and a low incidence of adverse side effects. As aresult of that research, they found that oligonucleotide analogues ornucleoside analogues having an ether bond in said molecules are usefulas an antisense or antigene pharmaceutical having excellent stability, adetection agent (probe) for a specific gene, a primer for startingamplification or as intermediates for their production, and accomplishedthe present invention.

In the following, the present invention will be described in detail.

The novel nucleoside analogues of the present invention are compounds ofthe formula (1):

wherein R¹ and R² are the same or different and represent a hydrogenatom, a hydroxyl protecting group, a phosphate group, a protectedphosphate group or —P(R³)R⁴ wherein R³ and R⁴ are the same or differentand represent a hydroxyl group, a protected hydroxyl group, a mercaptogroup, a protected mercapto group, an amino group, an alkoxy grouphaving from 1 to 4 carbon atoms, an alkylthio group having from 1 to 4carbon atoms, a cyanoalkoxy group having from 1 to 5 carbon atoms or anamino group substituted by an alkyl group having from 1 to 4 carbonatoms;

-   A represents an alkylene group having from 1 to 4 carbon atoms; and-   B represents an unsubstituted purin-9-yl group, an unsubstituted    2-oxo-pyrimidin-1-yl group or a substituted purin-9-yl group or a    substituted 2-oxo-pyrimidin-1-yl group having a substituent selected    from the α group defined hereinbelow;    or salts thereof.

The oligonucleotide analogues of the present invention areoligonucleotide analogues having two or more nucleoside units, whereinat least one of the nucleoside units is a structure of the formula (2):

wherein A represents an alkylene group having from 1 to 4 carbon atoms;and B represents an unsubstituted purin-9-yl group, an unsubstituted2-oxo-pyrimidin-1-yl group or a substituted purin-9-yl group or asubstituted 2-oxo-pyrimidin-1-yl group having a substituent selectedfrom the following α group;

-   α group:    -   an unprotected hydroxyl group,    -   a protected hydroxyl group,    -   an alkoxy group having from 1 to 4 carbon atoms,    -   an unprotected mercapto group,    -   a protected mercapto group,    -   an alkylthio group having from 1 to 4 carbon atoms,    -   an unprotected amino group,    -   a protected amino group,    -   an amino group substituted by an alkyl group having from 1 to 4        carbon atoms,    -   an alkyl group having from 1 to 4 carbon atoms, and    -   a halogen atom;        or a salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

“The alkylene group having from 1 to 4 carbon atoms” of A in the aboveformula (1) or (2) may include methylene, ethylene, trimethylene andtetramethylene groups, preferably a methylene group.

The protecting group of “the hydroxyl protecting group” of R¹ and R² and“the protected hydroxyl group” of R³ and R⁴ or the α group in the aboveformula (1) or (2) refers to a protecting group which can be cleaved bya chemical method such as hydrogenolysis, decomposition, hydrolysis,electrolysis and photolysis or a biological method such as hydrolysis inthe human body, and such protecting groups may include “an aliphaticacyl group” such as an alkylcarbonyl group, e.g., formyl, acetyl,propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl,isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl,8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl,dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl,1-methylpentadecanoyl, 14-methylpentadecanoyl,13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexadecanoyl,octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, eicosanoyl andheneicosanoyl, a carboxylated alkylcarbonyl group, e.g., succinoyl,glutaroyl and adipoyl, a halogeno lower alkylcarbonyl group, e.g.,chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl, alower alkoxy lower alkylcarbonyl group, e.g., methoxyacetyl, and anunsaturated alkylcarbonyl group, e.g., (E)-2-methyl-2-butenoyl;

-   “an aromatic acyl group” such as an arylcarbonyl group, e.g.,    benzoyl, α-naphthoyl and β-naphthoyl, a halogenoarylcarbonyl group,    e.g., 2-bromobenzoyl and 4-chloro-benzoyl, a lower alkylated    arylcarbonyl group, e.g., 2,4,6-trimethylbenzoyl and 4-toluoyl, a    lower alkoxylated arylcarbonyl group, e.g., 4-anisoyl, a    carboxylated arylcarbonyl group, e.g., 2-carboxybenzoyl,    3-carboxybenzoyl and 4-carboxybenzoyl, a nitrated arylcarbonyl    group, e.g., 4-nitrobenzoyl and 2-nitrobenzoyl, a lower alkoxy    carbonylated arylcarbonyl group, e.g., 2-(methoxycarbonyl)benzoyl    and an arylated arylcarbonyl group, e.g., 4-phenylbenzoyl;-   “a tetrahydropyranyl group or a tetrahydrothiopyranyl group” such as    tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl,    4-methoxytetrahydropyran-4-yl, tetrahydrothiopyran-2-yl and    4-methoxytetrahydrothiopyran-4-yl;-   “a tetrahydropyranyl group or a tetrahydrothiofuranyl group” such as    tetrahydrofuran-2-yl and tetrahydrothiofuran-2-yl;-   “a silyl group” such as a tri-lower alkylsilyl group, e.g.,    trimethylsilyl, triethylsilyl, isopropyldimethylsilyl,    t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl    and triisopropylsilyl and a tri-lower alkylsilyl group substituted    by one or two aryl groups, e.g., diphenylmethylsilyl,    diphenylbutylsilyl, diphenylisopropylsilyl and    phenyldiisopropylsilyl;-   “a lower alkoxymethyl group” such as methoxymethyl,    1,1-dimethyl-1-methoxy-methyl, ethoxymethyl, propoxymethyl,    isopropoxymethyl, butoxymethyl and t-butoxymethyl;-   “a lower alkoxylated lower alkoxymethyl group” such as    2-methoxyethoxymethyl;-   “a halogeno lower alkoxymethyl group” such as    2,2,2-trichloroethoxymethyl and bis(2-chloroethoxy)methyl;-   “a lower alkoxylated ethyl group” such as 1-ethoxyethyl and    1-(isopropoxy)ethyl;-   “a halogenated ethyl group” such as 2,2,2-trichloroethyl;-   “a methyl group substituted by from 1 to 3 aryl groups” such as    benzyl, α-naphthyl-methyl, β-naphthylmethyl, diphenylmethyl,    triphenylmethyl, α-naphthyldiphenyl-methyl and 9-anthrylmethyl;-   “a methyl group substituted by from 1 to 3 aryl groups wherein said    aryl ring is substituted by a lower alkyl, lower alkoxy, halogen or    cyano group” such as 4-methyl-benzyl, 2,4,6-trimethylbenzyl,    3,4,5-trimethylbenzyl, 4-methoxybenzyl,    4-methoxy-phenyldiphenylmethyl, 4,4′-dimethoxytriphenylmethyl,    2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl and    4-cyanobenzyl;-   “a lower alkoxycarbonyl group” such as methoxycarbonyl,    ethoxycarbonyl, t-butoxycarbonyl and isobutoxycarbonyl;-   “a lower alkoxycarbonyl group substituted by halogen or a tri-lower    alkylsilyl group” such as 2,2,2-trichloroethoxycarbonyl and    2-trimethylsilylethoxycarbonyl;-   “an alkenyloxycarbonyl group” such as vinyloxycarbonyl and    allyloxycarbonyl; and-   “an aralkyloxycarbonyl group wherein said aryl ring may be    substituted by one or two lower alkoxy or nitro groups” such as    benzyloxycarbonyl,-   4-methoxybenzyloxy-carbonyl, 3,4-dim ethoxybenzyloxycarbonyl,-   2-nitrobenzyloxy-carbonyl and-   4-nitrobenzyloxycarbonyl.

“The hydroxyl protecting group” of R¹ and R² may preferably include “thealiphatic acyl group”, “the aromatic acyl group”, “the methyl groupsubstituted by from 1 to 3 aryl groups”, “the methyl group substitutedby from 1 to 3 aryl groups wherein said aryl ring is substituted by alower alkyl, lower alkoxy, halogen or cyano group” or “the silyl group”;more preferably an acetyl group, a benzoyl group, a benzyl group, ap-methoxybenzoyl group, a dimethoxytrityl group, a monomethoxytritylgroup or a tert-butyldiphenylsilyl group.

The protecting group of the “protected hydroxyl group” of R³ and R⁴ orthe α group may preferably include “the aliphatic acyl group” or “thearomatic acyl group”, more preferably a benzoyl group.

The protecting group of “the protected phosphate group” of R¹ and R² inthe above formula (1) represents a protecting group which can be cleavedby a chemical method such as hydrogenolysis, hydrolysis, electrolysisand photolysis and a biological method such as hydrolysis in the humanbody and such protecting groups may include “a lower alkyl group” suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl,tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl,1-ethyl-propyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl,2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl,1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl,2,3-dimethylbutyl and 2-ethylbutyl;

-   “a cyanated lower alkyl group” such as 2-cyanoethyl and    2-cyano-1,1-dimethylethyl;-   “an ethyl group substituted by a silyl group” such as    2-methyldiphenylsilylethyl, 2-trimethylsilylethyl and    2-triphenylsilylethyl;-   “a halogenated lower alkyl group” such as 2,2,2-trichloroethyl,    2,2,2-tribromoethyl, 2,2,2-trifluoroethyl and    2,2,2-trichloro-1,1-dimethylethyl;-   “a lower alkenyl group” such as ethenyl, 1-propenyl, 2-propenyl,    1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl,    2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl,    1-methyl-2-butenyl, 1-methyl-1-butenyl, 3-methyl-2-butenyl,    1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl,    2-methyl-3-butenyl, 1-ethyl-3-butenyl, 1-pentenyl, 2-pentenyl,    1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl,    1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl,    1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 1-hexenyl, 2-hexenyl,    3-hexenyl, 4-hexenyl and 5-hexenyl,-   “a cycloalkyl group” such as cyclopropyl, cyclobutyl, cyclopentyl,    cyclohexyl, cycloheptyl, norbornyl and adamantyl;-   “a cyanated lower alkenyl group” such as 2-cyanobutenyl;-   “an aralkyl group” such as benzyl, α-naphthylmethyl,    β-naphthylmethyl, indenylmethyl, phenanethrenylmethyl,    anthracenylmethyl, diphenylmethyl, triphenylmethyl, 1-phenethyl,    2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl,    2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl,    3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl,    4-phenylbutyl, 1-naphthylbutyl, 2-naphthylbutyl, 3-naphthylbutyl,    4-naphthylbutyl, 1-phenylpentyl, 2-phenylpentyl, 3-phenylpentyl,    4-phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2-naphthylpentyl,    3-naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl, 1-phenylhexyl,    2-phenylhexyl, 3-phenylhexyl, 4-phenylhexyl, 5-phenylhexyl,    6-phenylhexyl, 1-naphthylhexyl, 2-naphthylhexyl, 3-naphthylhexyl,    4-naphthylhexyl, 5-naphthylhexyl and 6-naphthylhexyl;-   “an aralkyl group wherein said aryl ring is substituted by a nitro    group or a halogen atom” such as 4-chlorobenzyl,    2-(4-nitrophenyl)ethyl, o-nitrobenzyl, 4-nitrobenzyl,    2,4-di-nitrobenzyl and 4-chloro-2-nitrobenzyl;-   “an aryl group” such as phenyl, indenyl, naphthyl, phenanthrenyl and    anthracenyl;-   and “an aryl group substituted by a lower alkyl group, a halogen    atom or a nitro group” such as 2-methylphenyl, 2,6-dimethylphenyl,    2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl,    2,5-dichlorophenyl, 2-bromophenyl, 4-nitrophenyl and    4-chloro-2-nitrophenyl;-   preferably “the lower alkyl group”, “the lower alkyl group    substituted by a cyano group”, “the aralkyl group” or “the aralkyl    group wherein said aryl ring is substituted by a nitro group or a    halogen atom”; more preferably a 2-cyanoethyl group, a    2,2,2-trichloroethyl group or a benzyl group.

“The alkoxy group having from 1 to 4 carbon atoms” of R³ and R⁴ or the αgroup in the above formula (1) or (2) may include methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy or tert-butoxy,preferably a methoxy or ethoxy group.

The protecting group of “the protected mercapto group” of R³ and R⁴ orthe α group in the above formula (1) or (2) may include, in addition tothe hydroxyl protecting groups mentioned above, “a group which forms adisulfide” such as an alkylthio group, e.g., methylthio, ethylthio,tert-butylthio and an aralkylthio group such as benzylthio, preferably“the aliphatic acyl group” or “the aromatic acyl group”, more preferablya benzoyl group.

“The alkylthio group having from 1 to 4 carbon atoms” of R³ and R⁴ orthe α group in the above formula (1) or (2) may include methylthio,ethylthio, propylthio, isopropylthio, butylthio, isobutylthio,s-butylthio and tert-butylthio, preferably a methylthio or ethylthiogroup.

The protecting group of “the protected amino group” of the α group inthe above formula (1) or (2) may include

-   “an aliphatic acyl group” such as an alkylcarbonyl group, e.g.,    formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl,    valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl,    8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl,    dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl,    1-methylpentadecanoyl, 14-methylpentadecanoyl,    13,13-dimethyl-tetradecanoyl, heptadecanoyl, 15-methylhexadecanoyl,    octadecanoyl, 1-methyl-heptadecanoyl, nonadecanoyl, eicosanoyl and    heneicosanoyl, a carboxylated alkylcarbonyl group, e.g., succinoyl,    glutaroyl and adipoyl, a halogeno lower alkylcarbonyl group, e.g.,    chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl, a    lower alkoxy lower alkylcarbonyl group, e.g., methoxyacetyl, and an    unsaturated alkylcarbonyl group, e.g., (E)-2-methyl-2-butenoyl;-   “an aromatic acyl group” such as an arylcarbonyl group, e.g.,    benzoyl, α-naphthoyl and β-naphthoyl, a halogenoarylcarbonyl group,    e.g., 2-bromobenzoyl and 4-chlorobenzoyl, a lower alkylated    arylcarbonyl group, e.g., 2,4,6-trimethylbenzoyl and 4-toluoyl, a    lower alkoxylated arylcarbonyl group, e.g., 4-anisoyl, a    carboxylated arylcarbonyl group, e.g., 2-carboxybenzoyl,    3-carboxybenzoyl and 4-carboxybenzoyl, a nitrated arylcarbonyl    group, e.g., 4-nitrobenzoyl and 2-nitrobenzoyl, a lower alkoxy    carbonylated arylcarbonyl group, e.g., 2-(methoxycarbonyl)benzoyl    and an arylated arylcarbonyl group, e.g., 4-phenylbenzoyl;-   “a lower alkoxycarbonyl group” such as methoxycarbonyl,    ethoxycarbonyl, t-butoxycarbonyl and isobutoxycarbonyl;-   “a lower alkoxycarbonyl group substituted by halogen or a tri-lower    alkylsilyl group” such as 2,2,2-trichloroethoxycarbonyl and    2-trimethylsilylethoxycarbonyl;-   “an alkenyloxycarbonyl group” such as vinyloxycarbonyl and    allyloxycarbonyl; and-   “an aralkyloxycarbonyl group wherein said aryl ring may be    substituted by a lower alkoxy or nitro group” such as    benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,    3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, and    4-nitrobenzyloxy-carbonyl, preferably “the aliphatic acyl group or    “the aromatic acyl group”, more preferably a benzoyl group.

“The amino group substituted by an alkyl group having from 1 to 4 carbonatoms” of R³ and R⁴ or the α group in the above formula (1) or (2) mayinclude methylamino, ethylamino, propylamino, isopropylamino,butylamino, isobutylamino, s-butylamino, tert-butylamino, dimethylamino,diethylamino, dipropylamino, diisopropylamino, dibutylamino,diisobutylamino, di(s-butyl)amino and di(tert-butyl)amino, preferablymethylamino, ethylamino, dimethylamino, diethylamino ordiisopropylamino.

“The cyanoalkoxy group having from 1 to 5 carbon atoms” of R³ and R⁴ inthe above formula (1) represents a group in which the above-described“the alkoxy group having from 1 to 4 carbon atoms” is substituted by acyano group, and such a group may include cyanomethoxy, 2-cyanoethoxy,3-cyanopropoxy, 4-cyanobutoxy, 3-cyano-2-methylpropoxy or1-cyanomethyl-1,1-dimethylmethoxy, preferably a 2-cyanoethoxy group.

“The alkyl group having from 1 to 4 carbon atoms” of the α group in theabove formula (1) or (2) may include methyl, ethyl, propyl, isopropyl,butyl, isobutyl, s-butyl and tert-butyl, preferably a methyl or ethylgroup.

“The halogen atom” of the α group in the above formula (1) or (2) mayinclude a fluorine atom, a chlorine atom, a bromine atom or an iodineatom, preferably a fluorine atom or a chlorine atom.

The preferred groups of “the purin-9-yl group” and “the substitutedpurin-9-yl group” of B in the above formula (1) or (2) may include, as awhole, 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl the aminogroup of which is protected, 2,6-diaminopurin-9-yl,2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl the amino groupof which is protected, 2-amino-6-fluoropurin-9-yl,2-amino-6-fluoropurin-9-yl the amino group of which is protected,2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl the amino group ofwhich is protected, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl),2-amino-6-hydroxypurin-9-yl the amino group of which is protected,2-amino-6-hydroxypurin-9-yl the amino and hydroxyl groups of which areprotected, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl,6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl,2,6-dichloropurin-9-yl or 6-mercaptopurin-9-yl, more preferably a6-benzoylaminopurin-9-yl, adeninyl,2-isobutyrylamino-6-hydroxypurin-9-yl or guaninyl group.

The preferred groups of “the 2-oxo-pyrimidin-1-yl group” and “thesubstituted 2-oxo-pyrimidin-1-yl group” of B in the above formula (1) or(2) may include, as a whole, 2-oxo-4-amino-pyrimidin-1-yl (i.e.,cytosinyl), 2-oxo-4-amino-pyrimidin-1-yl the amino group of which isprotected, 2-oxo-4-amino-5-fluoro-pyrimidin-1-yl,2-oxo-4-amino-5-fluoro-pyrimidin-1-yl the amino group of which isprotected, 4-amino-2-oxo-5-chloro-pyrimidin-1-yl,2-oxo-4-methoxy-pyrimidin-1-yl, 2-oxo-4-mercapto-pyrimidin-1-yl,2-oxo-4-hydroxy-pyrimidin-1-yl (i.e., uracinyl),2-oxo-4-hydroxy-5-methylpyrimidin-1-yl (i.e., thyminyl) or4-amino-5-methyl-2-oxo-pyrimidin-1-yl (i.e., 5-methylcytosinyl) group,more preferably 2-oxo-4-benzoylamino-pyrimidin-1-yl, cytosinyl,thyminyl, uracinyl, 2-oxo-4-benzoylamino-5-methyl-pyrimidin-1-yl or5-methylcytosinyl group.

“The nucleoside analogue” refers to a non-natural type of “nucleoside”in which a purine or pyrimidine group is attached to a sugar.

“The oligonucleotide analogue” refers to a non-natural type of“oligonucleotide” derivative in which from 2 or more and up to 100 andpreferably 2 to 50 and more preferably 10 to 30 “nucleosides”, which maybe the same or different, are bonded through a phosphodiester bond andsuch analogues may preferably include sugar derivatives in which thesugar moiety is modified; thioate derivatives in which thephosphodiester bond moiety is thioated (phosphorothioate bond); esterproducts in which a terminal phosphate moiety is esterified; and amideproducts in which an amino group on a purine base is amidated, morepreferably the sugar derivatives in which the sugar moiety (ribose ordeoxyribose) is modified and the thioate derivatives in which thephosphodiester moiety is thioated.

Naturally occurring oligonucleotides are those which occur in nature,for example, ribose and deoxyribose phosphodiester oligonucleotideshaving adenine, guanine, cytosine, thymine and uracil nucleobases. Asused herein, “oligonucleotide analogues” are oligonucleotides thatcontain modified sugar, internucleoside linkage and/or nucleobasemoieties. Such oligonucleotide analogs are typically structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic wild type oligonucleotides. Thus, non-naturallyoccurring oligonucleotides include all such structures which functioneffectively to mimic the structure and/or function of a desired RNA orDNA strand, for example, by hybridizing to a target.

The nucleosides other than formula (2) in the oligonucleotide analoguesof the present invention are any of the known nucleosides or not yetknown nucleosides that are functionally interchangeable withnaturally-occurring nucleosides. Preferably such nucleosides have thestructure of a nucleobase and a sugar defined as follows.

Representative nucleobases include adenine, guanine, cytosine, uracil,and thymine, as well as other non-naturally occurring and naturalnucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl andother alkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil,8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substitutedadenines and guanines, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine. Further naturally and non naturallyoccurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808(Merigan, et al.), in chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, and Cook, Anti-Cancer DrugDesign, 1991, 6, 585-607, each of which publications are herebyincorporated by reference in their entirety). The term “nucleosidicbase” is further intended to include heterocyclic compounds that canserve as like nucleosidic bases including certain “universal bases” thatare not nucleosidic bases in the most classical sense but serve asnucleosidic bases. Especially mentioned as a universal base is3-nitropyrrole.

Preferred 2′-groups of the sugar include H, OH, F, and O—, S—, orN-alkyl groups. One particularly preferred group includes2′-methoxyethoxy[2′-O—CH₂ CH₂ OCH₃, also known as 2′-O-(2-methoxyethyl)or 2′-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486), i.e., analkoxyalkoxy group. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ ON(CH₃)₂ group, also known as2′-DMAOE, as described in U.S. Pat. No. 6,127,533, the entire contentsof which are herein incorporated by reference. Other preferredmodifications include 2′-methoxy (2′-O—CH₃) and 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂).

Sugars of nucleosides having O-substitutions on the ribosyl ring arealso amenable to the present invention. Representative substitutions forring O include S, CH₂, CHF, and CF₂, see, e.g., Secrist, et al.,Abstract 21, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sep. 16-20, 1992, which is hereby incorporated by reference in itsentirety.

Internucleoside linkages may be any of the known internucleosidelinkages, or may be any internucleoside linkage not yet known that canbe incorporated into an oligonucleotide according to synthetic chemistrywith which the process according to the invention is compatible. Incertain preferred embodiments, the other internucleoside linkages arephosphodiester or phosphorothioate linkages. In the case ofphosphorothioate internucleoside linkages, the linkages may bephosphorothioate mixed enantiomers or stereoregular phosphorothioates(see Iyer et al., Tetrahedron Asymmetry 6: 1051-1054 (1995).

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar, on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the oligonucleotides of thepresent invention involves chemically linking to the oligonucleotide oneor more moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide. Such moietiesinclude but are not limited to lipid moieties such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553),cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053),a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y.Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Non-limiting examples of nucleosides other than of the formula (2) areas follows: adenosine, guanosine, cytidine, 5-methylcytidine, uridine,5-methyluridine, inosine, 5-(1-propynyl)cytidine, 5-(1-propynyl)uridine,2′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxycytidine,5-methyl-2′-deoxycytidine, 2′-deoxyuridine,thymidine, 2′-deoxyinosine,2′-deoxy-5-(1-propynyl)cytidine, 2′-deoxy-5-(1-propynyl)uridine,2′-O-methyladenosine, 2′-O-methylguanosine, 2′-O-methylcytidine,5-methyl-2′-O-methylcytidine,2′-O-methyluridine,5-methyl-2′-O-methyluridine, 2′-O-methylinosine,5-(1-propynyl)-2′-O-methylcytidine, 5-(1-propynyl)-2′-O-methyluridine,2′-O-allyladenosine, 2′-O-allylguanosine, 2′-O-allylcytidine,5-methyl-2′-O-allylcytidine,2′-O-allyluridine,5-methyl-2′-O-allyluridine, 2′-O-allylinosine,5-(1-propynyl)-2′-O-allylcytidine, 5-(1-propynyl)-2′-O-allyluridine,2′-O-propargyladenosine, 2′-O-propargylguanosine,2′-O-propargylcytidine, 5-methyl-2′-O-propargylcytidine,2′-O-propargyluridine,5-methyl-2′-O-propargyluridine,2′-O-propargyllinosine, 5-(1-propynyl)-2′-O-propargylcytidine,5-(1-propynyl)-2′-O-allyluridine, 2′-O-(2-methoxyethyl)adenosine,2′-O-(2-methoxyethyl)guanosine, 2′-O-(2-methoxyethyl)cytidine,5-methyl-2′-O-(2-methoxyethyl)cytidine,2′-O-(2-methoxyethyl)uridine,5-methyl-2′-O-(2-methoxyethyl)uridine,2′-O-(2-methoxyethyl)inosine,5-(1-propynyl)-2′-O-(2-methoxyethyl)cytidine,5-(1-propynyl)-2′-O-(2-methoxyethyl)uridine,2′-O-(2-dimethylaminooxyethyl)adenosine,2′-O-(2-dimethylaminooxyethyl)guanosine,2′-O-(2-dimethylaminooxyethyl)cytidine,5-methyl-2′-O-(2-dimethylaminooxyethyl)cytidine,2′-O-(2-dimethylaminooxyethyl)uridine,5-methyl-2′-O-(2-dimethylaminooxyethyl)uridine, 2′-O-(2-dimethylaminooxyethyl)inosine,5-(1-propynyl)-2′-O-(2-dimethylaminooxyethyl)cytidine,5-(1-propynyl)-2′-O-(2-dimethylaminooxyethyl)uridine,2′-fluoro-2′-deoxyadenosine, 2′-fluoro-2′-deoxyguanosine,2′-fluoro-2′-deoxycytidine, 5-methyl-2′-fluoro-2′-deoxycytidine,2′-fluoro-2′-deoxyuridine, 5-methyl-2′-fluoro-2′-deoxyuridine,2′-fluoro-2′-deoxyinosine, 5-(1-propynyl)-2′-fluoro-2′-deoxyuridine,5-(1-propynyl)-2′-fluoro-2′-deoxyuridine, 2′-amino-2′-deoxyadenosine,2′-amino-2′-deoxyguanosine, 2′-amino-2′-deoxycytidine,5-methyl-2′-amino-2′-deoxycytidine,2′-amino-2′-deoxyuridine,5-methyl-2′-amino-2′-deoxyuridine,2′-amino-2′-deoxyinosine, 5-(1-propynyl)-2′-amino-2′-deoxyuridine, and5-(1-propynyl)-2′-amino-2′-deoxyuridine.

In some preferred embodiments of the oligonucleotide analogues accordingto the present invention, several adjacent oligonucleotide analoguescomprise two regions, which are the first and the second regions.Hereinafter “the first region” comprises one or more nucleosideanalogues of the formula (2) and each nucleoside is connected by aphosphodiester bond; hereinafter the “second region” comprises one ormore of a 2′-deoxynucleoside (e.g., 2′-deoxyadenosine,2′-deoxyguanosine, 2′-deoxycytidine, thymidine, 2′-deoxyuridine,5-substituted-2′-deoxycytidine or 5-substituted-2′-deoxyuridine) andeach nucleoside is connected by a phosphodiester bond or aphosphorothioate bond.

In certain particularly preferred oligonucleotide analogues, the totalnumber of nucleosides is from 5 to 100, more preferably 10 to 50, andthe oligonucleotide analogues comprise the second region whose number ofnucleoside residues is about half of the total number of nucleosideresidues flanked on both sides by the first region, whose number ofnucleoside is about a quarter of the total number of nucleosideresidues. In this case, each nucleoside of the second region ispreferably connected by a phosphorothioate bond and the bonds betweenthe first region and the second region are phosphodiester bonds orphosphorothioate bonds.

In other certain particularly preferred oligonucleotide analogues, thetotal number of nucleosides is from 5 to 100, and the entireoligonucleotide analogue comprises (a) one or more of the nucleosideanalogues of the formula (2) and one or more nucleosides selected fromthe group consisting of (b) a 2′-deoxynucleoside (e.g.2′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxycytidine, thymidine,2′-deoxyuridine, 5-substituted-2′-deoxycytidine or5-substituted-2′-deoxyuridine) and (c) a 2′-O-methyl ribonucleoside(e.g., 2′-O-methyladenosine, 2′-O-methylguanosine, 2′-O-methylcytidine,5-methyl-2′-O-methyluridine, 2′-O-methyluridine,5-substituted-2′-O-methylcytidine or 5-substituted-2′-O-methyluridine).In this case, each every other nucleoside is a nucleoside analogue ofthe formula (2) and the bonds between each nucleoside are preferablyphosphodiester bonds.

“The salt thereof” refers to salts of the compound (1) of the presentinvention since they can be converted to salts and such salts maypreferably include inorganic salts for example metal salts such asalkali metal salts, e.g., sodium salts, potassium salts and lithiumsalts, alkaline earth metal salts, e.g., calcium salts and magnesiumsalts, aluminum salts, iron salts, zinc salts, copper salts, nickelsalts and cobalt salts; amine salts such as inorganic salts, e.g.,ammonium salts, organic salts, e.g., t-octylamine salts, dibenzylaminesalts, morpholine salts, glucosamine salts, phenylglycine alkyl estersalts, ethylenediamine salts, N-methylglucamine salts, guanidine salts,diethylamine salts, triethylamine salts, dicyclohexylamine salts,N,N′-dibenzylethylenediamine salts, chloroprocaine salts, procainesalts, diethanol amine salts, N-benzyl-phenethylamine salts, piperazinesalts, tetramethylammonium salts and a tris(hydroxymethyl)aminomethanesalts; inorganic acid salts such as hydrohalogenic acid salts, e.g.,hydrofluoric acid salts, hydrochloric acid salts, hydrobromic acid saltsand hydroiodic acid salts, nitric acid salts, perchloric acid salts,sulfuric acid salts and phosphoric acid salts; organic acid salts suchas lower alkanesulfonic acid salts, e.g., methanesulfonic acid salts,trifluoromethanesulfonic acid salts and ethanesulfonic acid salts,arylsulfonic acid salts, e.g., benzenesulfonic acid salts andp-toluenesulfonic acid salts, acetic acid salts, malic acid salts,fumaric acid salts, succinic acid salts, citric acid salts, tartaricacid salts, oxalic acid salts and maleic acid salts; and amino acidsalts such as glycine salts, lysine salts, arginine salts, ornithinesalts, glutamic acid salts and aspartic acid salts.

The modified oligonucleotides or the polynucleotide analogues of thepresent invention can be converted to a salt. Where the modifiedoligonucleotides or polynucleotide analogues are to be used as a probe,a primer for starting amplification or as intermediates, the salts areany of the salts noted above for the salts of the compound (1) of thepresent invention. Where, however, they are to be used as apharmaceutical, the salts should be “pharmacologically acceptable saltsthereof”. The “pharmacologically acceptable salts thereof” refers to asalt thereof, and such salts may preferably include inorganic salts forexample metal salts such as alkali metal salts, e.g., sodium salts,potassium salts lithium salts, alkaline earth metal salts, e.g., calciumsalts and magnesium salts, aluminum salts, iron salts, zinc salts,copper salts, nickel salts and cobalt salts; amine salts such asinorganic salts, e.g., ammonium salts, organic salts, e.g., t-octylaminesalts, dibenzylamine salts, morpholine salts, glucosamine salts,phenylglycine alkyl ester salts, ethylenediamine salts,N-methylglucamine salts, guanidine salts, diethylamine salts,triethylamine salts, dicyclohexylamine salts,N,N′-dibenzylethylenediamine salts, chloroprocaine salts, procainesalts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazinesalts, tetra-methylammonium salts and tris(hydroxymethyl)aminomethanesalts; inorganic acid salts such as hydrohalogenic acid salts, e.g.,hydrofluoric acid salts, hydrochloric acid salts, hydrobromic acid saltsand hydroiodic acid salts, nitric acid salts, perchloric acid salts,sulfuric acid salts and phosphoric acid salts; organic acid salts suchas lower alkanesulfonic acid salts, e.g., methanesulfonic acid salts,trifluoromethanesulfonic acid salts and ethanesulfonic acid salts,arylsulfonic acid salts, e.g., benzenesulfonic acid salts andp-toluenesulfonic acid salts, acetic acid salts, malic acid salts,fumaric acid salts, succinic acid salts, citric acid salts, tartaricacid salts, oxalic acid salts and maleic acid salts; and amino acidsalts such as glycine salts, lysine salts, arginine salts, ornithinesalts, glutamic acid salts and aspartic acid salts.

The term “strand displacement” relates to a process whereby anoligonucleotide binds to its complementary target sequence in a doublestranded DNA or RNA so as to displace the other strand from said targetstrand

Several diagnostic and molecular biology procedures have been developedthat utilize panels of different oligonucleotides to simultaneouslyanalyze a target nucleic acid for the presence of a plethora of possiblemutations. Typically, the oligonucleotide panels are immobilized in apredetermined pattern on a solid support such that the presence of aparticular mutation in the target nucleic acid can be revealed by theposition on the solid support where it hybridizes. One importantprerequisite for the successful use of panels of differentoligonucleotides in the analysis of nucleic acids is that they are allspecific for their particular target sequence under the single appliedhybridization condition. Since the affinity and specificity of standardoligonucleotides for their complementary target sequences depend heavilyon their sequence and size this criteria has been difficult to fulfillso far.

In a preferred embodiment of the present invention, therefore,oligonucleotide analogues are used as a means to increase affinityand/or specificity of the probes and as a means to equalize the affinityof different oligonucleotides for their complementary sequences. Asdisclosed herein such affinity modulation can be accomplished by, e.g.,replacing selected nucleosides in the oligonucleotide with an nucleosideof formula (2) carrying a similar nucleobase.

In another preferred embodiment of the present invention, the highaffinity and specificity of oligonucleotide analogues is exploited inthe sequence specific capture and purification of natural or syntheticnucleic acids. In one aspect, the natural or synthetic nucleic acids arecontacted with oligonucleotide analogues immobilized on a solid surface.In this case hybridization and capture occurs simultaneously. Thecaptured nucleic acids may be, for instance, detected, characterized,quantified or amplified directly on the surface by a variety of methodswell known in the art or it may be released from the surface, beforesuch characterization or amplification occurs, by subjecting theimmobilized, modified oligonucleotide and captured nucleic acid todehybridizing conditions, such as, for example, heat or by using buffersof low ionic strength.

The solid support may be chosen from a wide range of polymer materialssuch as, for instance, CPG (controlled pore glass), polypropylene,polystyrene, polycarbonate or polyethylene and it may take a variety offorms such as, for instance, a tube, a microtiter plate, a stick, abead, a filter, etc. The oligonucleotide analogues may be immobilized tothe solid support via its 5′ or 3′ end (or via the terminus of linkersattached to the 5′ or 3′ end) by a variety of chemical or photochemicalmethods usually employed in the immobilization of oligonucleotides or bynon-covalent coupling such as for instance via binding of a biotinylatedoligonucleotide analogues to immobilized streptavidin. One preferredmethod for immobilizing oligonucleotide analogues on different solidsupports is a photochemical method using a photochemically activeanthraquinone covalently attached to the 5′ or 3′ end of theoligonucleotide analogues (optionally via linkers) as described in WO96/31557. Thus, the present invention also provides a surface carryingan oligonucleotide analogue.

In another aspect, the oligonucleotide analogue carries a ligandcovalently attached to either the 5′ or 3′ end. In this case theoligonucleotide analogue is contacted with natural or synthetic nucleicacids in solution whereafter the hybrids formed are captured onto asolid support carrying molecules that can specifically bind the ligand.

In still another aspect, oligonucleotide analogues capable of performing“strand displacement” are used in the capture of natural and syntheticnucleic acids without prior denaturation. Such modified oligonucleotidesare particularly useful in cases where the target sequence is difficultor impossible to access by normal oligonucleotides due to the rapidformation of stable intramolecular structures.

Examples of nucleic acids containing such structures are rRNA, tRNA,snRNA and scRNA.

In another preferred embodiment of the present invention,oligonucleotide analogues designed with the purpose of high specificityare used as primers in the sequencing of nucleic acids and as primers inany of the several well known amplification reactions, such as the PCRreaction. As shown herein, the design of the oligonucleotide analoguesdetermines whether it will sustain a exponential or linear targetamplification. The products of the amplification reaction can beanalyzed by a variety of methods applicable to the analysis ofamplification products generated with normal DNA primers. In theparticular case where the oligonucleotide analogue primers are designedto sustain a linear amplification the resulting amplicons will carrysingle stranded ends that can be targeted by complementary probeswithout denaturation.

Such ends could for instance be used to capture amplicons by othercomplementary oligonucleotide analogues attached to a solid surface.

In another aspect, oligonucleotide analogues capable of “stranddisplacement” are used as primers in either linear or exponentialamplification reactions. The use of such oligos is expected to enhanceoverall amplicon yields by effectively competing with ampliconre-hybridization in the later stages of the amplification reaction.Demers, et al. (Nucl. Acid Res., 1995, Vol 23, 3050-3055) discloses theuse of high-affinity, non-extendible oligos as a means of increasing theoverall yield of a PCR reaction. It is believed that the oligomerselicit these effect by interfering with amplicon re-hybridization in thelater stages of the PCR reaction. It is expected that oligonucleotideanalogue blocked at their 3′ end will provide the same advantage.Blocking of the 3′ end can be achieved in numerous ways like forinstance by exchanging the 3′ hydroxyl group with hydrogen or phosphate.Such 3′ blocked oligonucleotide analogues can also be used toselectively amplify closely related nucleic acid sequences in a waysimilar to that described by Yu et al. (Biotechniques, 1997, 23,714-716).

In recent years, novel classes of probes that can be used in, forexample, real-time detection of amplicons generated by targetamplification reactions have been invented.

One such class of probes have been termed “Molecular Beacons”. Theseprobes are synthesized as partly self-complementary oligonucleotidescontaining a fluorophor at one end and a quencher molecule at the otherend. When free in solution, the probe folds up into a hairpin structure(guided by the self-complimentary regions) which positions the quencherin sufficient closeness to the fluorophor to quench its fluorescentsignal. Upon hybridization to its target nucleic acid, the hairpin opensthereby separating the fluorophor and quencher and giving off afluorescent signal.

Another class of probes have been termed “Taqman probes”. These probesalso contain a fluorophor and a quencher molecule. Contrary to the“Molecular Beacons”, however, the ability of the quenchers to quench thefluorescent signal from the fluorophor is maintained after hybridizationof the probe to its target sequence. Instead, the fluorescent signal isgenerated after hybridization by physical detachment of either thequencher or the fluorophor from the probe by the action of the 5′exonuclease activity of a polymerase which has initiated synthesis froma primer located 5′ to the binding site of the Taqman probe.

High affinity for the target site is an important feature in both typesof probes and consequently such probes tends to be fairly large(typically 30 to 40 mers). As a result, significant problems areencountered in the production of high quality probes.

In a preferred embodiment, therefore, the oligonucleotide analogue isused to improve production and subsequent performance of “Taqman probes”and “Molecular Beacons” by reducing their size, whilst retaining therequired affinity.

In a further aspect, the oligonucleotide analogues are used to constructnew affinity pairs (either fully or partially modifiedoligonucleotides). The affinity constants can easily be adjusted over awide range and a vast number of affinity pairs can be designed andsynthesized.

One part of the affinity pair can be attached to the molecule ofinterest (e.g., proteins, amplicons, enzymes, polysaccharides,antibodies, haptens, peptides, PNA, etc.) by standard methods, while theother part of the affinity pair can be attached to e.g., a solid supportsuch as beads, membranes, microliter plates, sticks, tubes, etc. Thesolid support may be chosen from a wide range of polymer materials suchas for instance polypropylene, polystyrene, polycarbonate orpolyethylene. The affinity pairs may be used in selective isolation,purification, capture and detection of a diversity of the targetmolecules mentioned above.

The principle of capturing oligonucleotide analogue by ways ofinteraction with another complementary oligonucleotide analogue (eitherfully or partially modified) can be used to create an infinite number ofnovel affinity pairs.

In another preferred embodiment, the high affinity and specificity ofthe oligonucleotide analogues are exploited in the construction ofprobes useful in in-situ hybridization. For instance, an oligonucleotideanalogue could be used to reduce the size of traditional DNA probes,whilst maintaining the required affinity thereby increasing the kineticsof the probe and its ability to penetrate the sample specimen. Theability of the oligonucleotide analogues to “strand displace” doublestranded nucleic acid structures are also of considerable advantage inin-situ hybridization, because it facilitates hybridization withoutprior denaturation of the target DNA/RNA.

The present invention also provides a kit for the isolation,purification, amplification, detection, identification, quantification,or capture of natural or synthetic nucleic acids, wherein the kitcomprises a reaction body and one or more oligonucleotide analogues asdefined herein. The oligonucleotide analogues are preferably immobilizedonto said reaction body (e.g., by using the immobilizing techniquesdescribed above).

For the kits according to the invention, the reaction body is preferablya solid support material, e.g., selected from borosilicate glass,soda-lime glass, polystyrene, polycarbonate, polypropylene,polyethylene, polyethyleneglycol terephthalate, polyvinyl acetate,polyvinylpyrrolidinone, polymethylmethacrylate and polyvinylchloride,preferably polystyrene and polycarbonate. The reaction body may be inthe form of a specimen tube, a vial, a slide, a sheet, a film, a bead, apellet, a disc, a plate, a ring, a rod, a net, a filter, a tray, amicrotiter plate, a stick, or a multi-bladed stick.

The kits are typically accompanied by a written instruction sheetstating the optimal conditions for the use of the kit.

“Antigene activity” is the ability to inhibit gene expression by forminga triplex with a DNA duplex. “Antisense activity” is the ability toinhibit gene expression by forming a duplex with a sense sequence. Atriplex with a DNA duplex means the state that an oligonucleotide fitsinto the groove of a DNA duplex strand, known as a “major groove”.

The oligonucleotides of the present invention can be used indiagnostics, therapeutics and as research reagents and kits. They can beused in pharmaceutical compositions by including a suitablepharmaceutically acceptable diluent or carrier. They further can be usedfor treating organisms having a disease characterized by the undesiredproduction of a protein. The organism should be contacted with anoligonucleotide having a sequence that is capable of specificallyhybridizing with a strand of nucleic acid coding for the undesirableprotein. Treatments of this type can be practiced on a variety oforganisms ranging from unicellular prokaryotic and eukaryotic organismsto multicellular eukaryotic organisms. Any organism that utilizesDNA-RNA transcription or RNA-protein translation as a fundamental partof its hereditary, metabolic or cellular control is susceptible totherapeutic and/or prophylactic treatment in accordance with the presentinvention. Seemingly diverse organisms such as bacteria, yeast,protozoa, algae, all plants and all higher animal forms, includingwarm-blooded animals, including humans, can be treated. Further, eachcell of multicellular eukaryotes can be treated, as they include bothDNA-RNA transcription and RNA-protein translation as integral parts oftheir cellular activity. Furthermore, many of the organelles (e.g.,mitochondria and chloroplasts) of eukaryotic cells also includetranscription and translation mechanisms. Thus, single cells, cellularpopulations or organelles can also be included within the definition oforganisms that can be treated with therapeutic or diagnosticoligonucleotides.

Some representative therapeutic indications and other uses for thecompounds of the invention are as follows:

One of the most significant health problems is the inadequate treatmentof pain. The impact of pain places great burden in economic terms aswell as in human suffering. Neuropathic pain is one of the mostdifficult pains to treat and cure. The primary site of this abnormal andectopic site is the dorsal root ganglion (DRG) of the injured site. Inthe DRG, two main types of sodium currents, termed TTX-sensitive andTTX-resistant, have been identified. The blockage of the sodium channelPN3/SNS, which is TTX-resistant, is a candidate for pain relief.Antisense compounds targeted to PN3/SNS are described in Porreca et al.Proc. Natl. Acad. Sci. 1999, 96, 7640-7644.

Another therapeutic indication of particular interest with respect tothe present invention is psoriasis. Psoriasis is a common chronic andrecurrent disease characterized by dry, well-circumscribed, silvery,scaling papules and plaques of various sizes. The disease varies inseverity from a few lesions to widespread dermatosis with disablingarthritis or exfoliation. The ultimate cause of psoriasis is presentlynot known, but the thick scaling that occurs is probably due toincreased epidermal cell proliferation (The Merck Manual of Diagnosisand Therapy, 15th Ed., pp. 2283-2285, Berkow et al., eds., Rahway, N.J.,1987). Inhibitors of Protein Kinase C(PKC) have been shown to have bothantiproliferative and anti-inflammatory effects in vitro. Someantipsoriasis drugs, such as cyclosporin A and anthralin, have beenshown to inhibit PKC, and inhibition of PKC has been suggested as atherapeutic approach to the treatment of psoriasis (Hegemann, L. and G.Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., pp. 357-368, CRCPress, Boca Raton, Fla., 1992). Antisense compounds targeted to ProteinKinase C(PKC) proteins are described in U.S. Pat. No. 5,620,963 to Cooket al. and U.S. Pat. No. 5,681,747 to Boggs et al.

A further therapeutic indication of interest to the present inventionare inflammatory disorders of the skin. These occur in a variety offorms including, for example, lichen planus, toxic epidermal necrolyis(TEN), ertythema multiforme and the like (The Merck Manual of Diagnosisand Therapy, 15th Ed., pp. 2286-2292, Berkow et al., eds., Rahway, N.J.,1987). Expression of ICAM-1 has been associated with a variety ofinflammatory skin disorders such as allergic contact dermatitis, fixeddrug eruption, lichen planus and psoriasis (Ho et al., J. Am. Acad.Dermatol., 1990, 22, 64; Griffiths et al., Am. J. Pathology, 1989, 135,1045; Lisby et al., Br. J. Dermatol., 1989, 120, 479; Shiohara et al.,Arch. Dermatol., 1989, 125, 1371; Regezi et al., Oral Surg. Oral Med.Oral Pathol., 1996, 81, 682). Moreover, intraperitoneal administrationof a monoclonal antibody to ICAM-1 decreases ovalbumin-inducedeosinophil infiltration into skin in mice (Hakugawa et al., J.Dermatol., 1997, 24, 73). Antisense compounds targeted to ICAM-1 aredescribed in U.S. Pat. Nos. 5,514,788, 5,591,623 and 6,111,094.

Other antisense targets for skin inflammatory disorders are VCAM-1 andPECAM-1. Intraperitoneal administration of a monoclonal antibody toVCAM-1 decreases ovalbumin-induced eosinophil infiltration into the skinof mice (Hakugawa et al., J. Dermatol., 1997, 24, 73). Antisensecompounds targeted to VCAM-1 are described in U.S. Pat. Nos. 5,514,788and 5,591,623. PECAM-1 proteins are glycoproteins which are expressed onthe surfaces of a variety of cell types (for reviews, see Newman, J.Clin. Invest., 1997, 99, 3 and DeLisser et al., Immunol. Today, 1994,15, 490). In addition to directly participating in cell-cellinteractions, PECAM-1 apparently also regulates the activity and/orexpression of other molecules involved in cellular interactions (Litwinet al., J. Cell Biol., 1997, 139, 219) and is thus a key mediator ofseveral cell:cell interactions. Antisense compounds targeted to PECAM-1are described in U.S. Pat. No. 5,955,443.

Another type of therapeutic indication of interest for usingoligonucleotides of the present invention encompasses a variety ofcancers of the skin. Representative skin cancers include benign tumors(warts, moles and the like) and malignant tumors such as, for example,basal cell carcinoma, squamous cell carcinoma, malignant melanoma,Paget's disease, Kaposi's sarcoma and the like (The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 2301-2310, Berkow et al., eds.,Rahway, N.J., 1987). A number of molecular targets involved intumorigenesis, maintenance of the hyperproliferative state andmetastasis are targeted to prevent or inhibit skin cancers, or toprevent their spread to other tissues.

The ras oncogenes are guanine-binding proteins that have been implicatedin cancer by, e.g., the fact that activated ras oncogenes have beenfound in about 30% of human tumors generally; this figure approached100% in carcinomas of the exocrine pancreas (for a review, see Downward,Trends in Biol. Sci., 1990, 15, 469). Antisense compounds targeted toH-ras and K-ras are described in U.S. Pat. No. 5,582,972 to Lima et al.,U.S. Pat. No. 5,582,986 to Monia et al. and U.S. Pat. No. 5,661,134 toCook et al., and in published PCT application WO 94/08003.

Protein Kinase C(PKC) proteins have also been implicated intumorigenesis. Antisense compounds targeted to Protein Kinase C(PKC)proteins are described in U.S. Pat. No. 5,620,963 to Cook et al. andU.S. Pat. No. 5,681,747 to Boggs et al. Also of interest are AP-1subunits and JNK proteins, particularly in regard to their roles intumorigenesis and metastasis. The process of metastasis involves asequence of events wherein (1) a cancer cell detaches from itsextracellular matrices, (2) the detached cancer cell migrates to anotherportion of an animal's body, often via the circulatory system, and (3)attaches to a distal and inappropriate extracellular matrix, therebycreated a focus from which a secondary tumor can arise. Normal cells donot possess the ability to invade or metastasize and/or undergoapoptosis (programmed cell death) if such events occur (Ruoslahti, Sci.Amer., 1996, 275, 72). However, many human tumors have elevated levelsof activity of one or more matrix metalloproteinases (MMPs)(Stetler-Stevenson et al., Annu. Rev. Cell Biol., 1993, 9, 541; Bernhardet al., Proc. Natl. Acad. Sci. (U.S.A.), 1994, 91, 4293. The MMPs are afamily of enzymes which have the ability to degrade components of theextracellular matrix (Birkedal-Hansen, Current Op. Biol., 1995, 7, 728).In particular, one member of this family, matrix metalloproteinase-9(MMP-9), is often found to be expressed only in tumors and otherdiseased tissues (Himelstein et al., Invasion & Metastasis, 1994, 14,246).

Several studies have shown that regulation of the MMP-9 gene may becontrolled by the AP-1 transcription factor (Kerr et al., Science, 1988,242, 1242; Kerr et al., Cell, 1990, 61, 267; Gum et al., J. Biol. Chem.,1996, 271, 10672; Hua et al., Cancer Res., 1996, 56, 5279). Inhibitionof AP-1 function has been shown to attenuate MMP-9 expression (U.S. Pat.No. 5,985,558). AP-1 is a heterodimeric protein having two subunits, thegene products of fos and jun. Antisense compounds targeted to c-fos andc-jun are described in U.S. Pat. No. 5,985,558.

Furthermore, AP-1 is itself activated in certain circumstances byphosphorylation of the Jun subunit at an amino-terminal position by JunN-terminal kinases (JNKs). Thus, inhibition of one or more JNKs isexpected to result in decreased AP-1 activity and, consequentially,reduced MMP expression. Antisense compounds targeted to JNKs aredescribed in U.S. Pat. No. 5,877,309.

Infectious diseases of the skin are caused by viral, bacterial or fungalagents. In the case of Lyme disease, the tick borne causative agentthereof, the spirochete Borrelia burgdorferi, up-regulates theexpression of ICAM-1, VCAM-1 and ELAM-1 on endothelial cells in vitro(Boggemeyer et al., Cell Adhes. Comm., 1994, 2, 145). Furthermore, ithas been proposed that the mediation of the disease by theanti-inflammatory agent prednisolone is due in part to mediation of thisup-regulation of adhesion molecules (Hurtenbach et al., Int. J.Immunopharmac., 1996, 18, 281). Thus, potential targets for therapeuticmediation (or prevention) of Lyme disease include ICAM-1, VCAM-1 andELAM-1 (supra).

Other infectious disease of the skin which are tractable to treatmentusing the compositions and methods of the invention include disordersresulting from infection by bacterial, viral or fungal agents (The MerckManual of Diagnosis and Therapy, 15th Ed., pp. 2263-2277, Berkow et al.,eds., Rahway, N.J., 1987).

With regard to infections of the skin caused by fungal agents, U.S. Pat.No. 5,691,461 describes antisense compounds for inhibiting the growth ofCandida albicans.

With regard to infections of the skin caused by viral agents, U.S. Pat.Nos. 5,166,195, 5,523,389 and 5,591,600 concern oligonucleotideinhibitors of Human Immunodeficiency Virus (HIV). U.S. Pat. No.5,004,810 is directed to oligomers capable of hybridizing to herpessimplex virus Vmw65 mRNA and inhibiting its replication. U.S. Pat. Nos.5,194,428 and 5,580,767 disclose antisense compounds having antiviralactivity against influenza virus. U.S. Pat. No. 4,806,463 relates toantisense compounds and methods using them to inhibit HTLV-IIIreplication. U.S. Pat. Nos. 4,689,320, 5,442,049, 5,591,720 and5,607,923 are directed to antisense compounds as antiviral agentsspecific to cytomegalovirus (CMV). U.S. Pat. No. 5,242,906 describesantisense compounds useful in the treatment of latent Epstein-Barr virus(EBV) infections. U.S. Pat. Nos. 5,248,670, 5, 514,577 and 5,658,891provide antisense compounds useful in the treatment of herpesvirusinfections. U.S. Pat. Nos. 5,457,189 and 5,681,944 disclose antisensecompounds useful in the treatment of papillomavirus infections. Theantisense compounds disclosed in the aforesaid U.S. patents, all ofwhich U.S. patents are herein incorporated by reference, may be usedwith (or replaced by) the compositions of the present invention toeffect prophylactic, palliative or therapeutic relief from diseasescaused or exacerbated by the indicated pathogenic agents.

Antisense oligonucleotides of the present invention may also be used todetermine the nature, function and potential relationship of variousgenetic components of the body to disease or body states in animals.Heretofore, the function of a gene has been chiefly examined by theconstruction of loss-of-function mutations in the gene (i.e.,“knock-out” mutations) in an animal (e.g., a transgenic mouse). Suchtasks are difficult, time-consuming and cannot be accomplished for genesessential to animal development, since the “knock-out” mutation wouldproduce a lethal phenotype. Moreover, the loss-of-function phenotypecannot be transiently introduced during a particular part of theanimal's life cycle or disease state; the “knock-out” mutation is alwayspresent. “Antisense knockouts,” that is, the selective modulation ofexpression of a gene by antisense oligonucleotides, rather than bydirect genetic manipulation, overcomes these limitations (see, forexample, Albert et al., Trends in Pharmacological Sciences, 1994, 15,250). In addition, some genes produce a variety of mRNA transcripts as aresult of processes such as alternative splicing; a “knock-out” mutationtypically removes all forms of mRNA transcripts produced from such genesand thus cannot be used to examine the biological role of a particularmRNA transcript. Antisense oligonucleotides have been systemicallyadministered to rats in order to study the role of theN-methyl-D-aspartate receptor in neuronal death, to mice in order toinvestigate the biological role of protein kinase C-a, and to rats inorder to examine the role of the neuropeptide Y1 receptor in anxiety(Wahlestedt et al.; Nature, 1993, 363:260; Dean et al., Proc. Natl.Acad. Sci. U.S.A., 1994, 91:11762; and Wahlestedt et al., Science, 1993,259:528, respectively). In instances where complex families of relatedproteins are being investigated, “antisense knockouts” (i.e., inhibitionof a gene by systemic administration of antisense oligonucleotides) mayrepresent the most accurate means for examining a specific member of thefamily (see, generally, Albert et al., Trends Pharmacol. Sci., 1994,15:250). By providing compositions and methods for the simplenon-parenteral delivery of oligonucleotides and other nucleic acids, thepresent invention overcomes these and other shortcomings.

With the growing insight of the potential biological role of triplehelical nucleic acids and the therapeutic potential ofoligonucleotide-directed triplex formation in the control of geneexpression according to the antigene strategy, research in triplehelical structures has been considerably stimulated. Thus, in theantigene approach, oligonucleotides are targeted to the unique gene thatspecifies a disease-related protein and stall transcription by bindingto the major groove of the doublestranded DNA target. Articles whichcontain a good review of this are Thuong & Mine in Angew. Chem. Int. Ed.Engl. 1993 32, pages 666-690 and “Prospects for the Therapeutic Use ofAntigene Oligonucleotides”, Maher, L. J. (1996) Cancer Investigation14(1), 66-82 each of which are hereby incorporated by reference in theirentirety.

A review of the development of the antigene strategies for designingdrugs that will bind to selected sites on the nucleic acids (DNA andRNA) is found in an article by J. S. Cohen and M. E. Hogan in ScientificAmerican, December 1994, pages 50-55 and in the monograph by Soyfer, V.N. & Potaman, V N. (1996). “Triple-helical nucleic acids”,Springer-Verlag, New York.

One of the diseases of interest as an antigene therapeutical target iscancer. The type I insulin-like growth factor receptor (IGF-IR) plays animportant role in the maintenance of the malignant phenotype of cancer(Rubin, R. & Baeserga, R. Lab. Invest. 73, 311 (1995)). A large numberof cancers and cancer-derived cell lines overexpress the IGF-IR(LeRoith, D et al, Endocr. Rev. 16, 143 (1995)). Antisense expressionvectors directed against the IGF-IR have proven effective in suppressingtumor growth of C6 rat glioblastoma (Baeserga, R. et al, Cancer Res. 54,2218 (1994)), hamster mesothelioma (Resnicoff, M. et al, Cancer Immunol.Immunother. 42, 64 (1996)), and rat prostate cancer (Pass, H. et al,Cancer Res. 56, 4044 (1996)). An antigene molecule expressed in rat C6glioblastoma cells inhibited IGF-I transcription and tumorigenicpotential of the cell (Rininsland, F. et al, Proc. Natl. Acad. Sci. USA94, 5854 (1997)). A compound inhibiting the expression of IGF-IR bymeans of antigene activity would be a medicament for the above describedtypes of cancer.

Antigene drugs can be used to treat the following diseases:

-   Anti-Virus

HIV (Giovannangeli, C. et al., Proc. Natl. Acad. Sci. USA, (1992) 89,8631-8635)

-   Anti-Cancer

human multidrug-resistance mdrl gene

(Morassutti, C. et al., Antisense Nucleic Acid Drug Dev, (1999) 9,261-270)

human HER-2/neu gene

(Ebbinghaus, S. W. et al., Biochemistry, (1999) 38, 619-628)

human c-myc gene

(Catapano, C. V. et al., Biochemistry, (2000) 39, 5126-5138)

Of the compounds (1) and the salts thereof of the present invention,preferred compounds include the following:

-   (1) compounds in which R¹ is a hydrogen atom, an aliphatic acyl    group, an aromatic acyl group, a methyl group substituted by from 1    to 3 aryl groups, a methyl group substituted by from 1 to 3 aryl    groups the aryl ring of which is substituted by a lower alkyl, lower    alkoxy, halogen or cyano group, or a silyl group, and salts thereof;-   (2) compounds in which R¹ is a hydrogen atom, an acetyl group, a    benzoyl group, a benzyl group, a p-methoxybenzyl group, a    dimethoxytrityl group, a mono-methoxytrityl group or a    tert-butyldiphenylsilyl group, and salts thereof;-   (3) compounds in which R² is a hydrogen atom, an aliphatic acyl    group, an aromatic acyl group, a methyl group substituted by from 1    to 3 aryl groups, a methyl group substituted by from 1 to 3 aryl    groups the aryl ring of which is substituted by a lower alkyl, lower    alkoxy, halogen or cyano group, a silyl group, a phosphoramidite    group, a phosphonyl group, a phosphate group or a protected    phosphate group, and salts thereof;-   (4) compounds in which R² is a hydrogen atom, an acetyl group, a    benzoyl group, a benzyl group, a p-methoxybenzyl group, a    tert-butyldiphenylsilyl group, —P(OC₂H₄CN)(NCH(CH₃)₂),    —P(OCH₃)(NCH(CH₃)₂), a phosphonyl group or a 2-chlorophenyl or    4-chlorophenyl phosphate group, and salts thereof;-   (5) compounds in which A is a methylene group, and salts thereof;-   (6) compounds in which B is a 6-aminopurin-9-yl (i.e., adeninyl),    6-aminopurin-9-yl the amino group of which is protected,    2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl,    2-amino-6-chloropurin-9-yl the amino group of which is protected,    2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl the amino    group of which is protected, 2-amino-6-bromopurin-9-yl,    2-amino-6-bromopurin-9-yl the amino group of which is protected,    2-amino-6-hydroxypurin-9-yl (i.e., guaninyl),    2-amino-6-hydroxypurin-9-yl the amino group of which is protected,    2-amino-6-hydroxypurin-9-yl the amino group and hydroxyl group of    which are protected, 6-amino-2-methoxypurin-9-yl,    6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl,    2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl,    6-mercaptopurin-9-yl, 2-oxo-4-amino-pyrimidin-1-yl (i.e.,    cytosinyl), 2-oxo-4-amino-pyrimidin-1-yl the amino group of which is    protected, 2-oxo-4-amino-5-fluoro-pyrimidin-1-yl,    2-oxo-4-amino-5-fluoro-pyrimidin-1-yl the amino group of which is    protected, 4-amino-2-oxo-5-chloro-pyrimidin-1-yl,    2-oxo-4-methoxy-pyrimidin-1-yl, 2-oxo-4-mercapto-pyrimidin-1-yl,    2-oxo-4-hydroxy-pyrimidin-1-yl (i.e., uracinyl),    2-oxo-4-hydroxy-5-methylpyrimidin-1-yl (i.e., thyminyl),    4-amino-5-methyl-2-oxo-pyrimidin-1-yl (i.e., 5-methylcytosinyl)    group or 4-amino-5-methyl-2-oxo-pyrimidin-1-yl group the amino of    which group is protected, and salts thereof; and-   (7) compounds in which B is a 6-benzoylaminopurin-9-yl, adeninyl,    2-isobutyrylamino-6-hydroxypurin-9-yl, guaninyl,    2-oxo-4-benzoylamino-pyrimidin-1-yl, cytosinyl,    2-oxo-5-methyl-4-benzoylamino-pyrimidin-1-yl, 5-methylcytosinyl,    uracinyl or thyminyl group, and salts thereof.

The above (1) and (2), (3) and (4) or (6) and (7) indicate the morepreferred compounds as the number becomes larger and in the formula (1),the compound obtained by optionally selecting R¹ from (1) and (2),optionally selecting R² from (3) and (4), optionally selecting A from(5) and optionally selecting B from (6) and (7) or by optionallycombining them and the salts thereof are preferred and the compounds andthe salts thereof selected from the following groups are particularlypreferred.

Group of Compounds:

-   2′-O,4′-C-ethyleneguanosine,-   2′-O,4′-C-ethyleneadenosine,-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-6-N-benzoyladenosine,-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-6-N-benzoyladenosine,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine,-   2′-O,4′-C-ethylene-2-N-isobutyrylguanosine,-   2′-O,4′-C-ethylene-6-N-benzoyladenosine,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-6-N-benzoyladenosine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite,-   2′-O,4′-C-ethyleneuridine,-   2′-O,4′-C-ethylene-5-methyluridine,-   2′-O,4′-C-ethylenecytidine,-   2′-O,4′-C-ethylene-5-methylcytidine,-   3′,5′-di-O-benzyl-2′-O,4′-C-ethyleneuridine,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethyleneuridine,-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-5-methyluridine,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-5-methyluridine,-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-4-N-benzoylcytidine,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoylcytidine,-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine,-   2′-O,4′-C-ethylene-4-N-benzoylcytidine,-   2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-uridine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-5-methyluridine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite,-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoylcytidine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite, and-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine-3′-O-(2-cyano    ethyl. N,N-diisopropyl)phosphoramidite.

Of the oligonucleotide analogues containing one or two or morestructures of the formula (2) and the salts thereof of the presentinvention, the preferred compounds may include

-   (8) oligonucleotide analogues in which A is a methylene group, and    pharmacologically acceptable salts thereof;-   (9) oligonucleotide analogues in which B is a 6-aminopurin-9-yl    (i.e., adeninyl), 6-aminopurin-9-yl the amino group of which is    protected, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl,    2-amino-6-chloropurin-9-yl the amino group of which is protected,    2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl the amino    group of which is protected, 2-amino-6-bromopurin-9-yl,    2-amino-6-bromopurin-9-yl the amino group of which is protected,    2-amino-6-hydroxypurin-9-yl (i.e., guaninyl),    2-amino-6-hydroxypurin-9-yl the amino group of which is protected,    2-amino-6-hydroxypurin-9-yl the amino group and hydroxyl group of    which are protected, 6-amino-2-methoxypurin-9-yl,    6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl,    2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl,    6-mercaptopurin-9-yl, 2-oxo-4-amino-pyrimidin-1-yl (i.e.,    cytosinyl), 2-oxo-4-amino-pyrimidin-1-yl the amino group of which is    protected, 2-oxo-4-amino-5-fluoro-pyrimidin-1-yl,    2-oxo-4-amino-5-fluoro-pyrimidin-1-yl the amino group of which is    protected, 4-amino-2-oxo-5-chloro-pyrimidin-1-yl,    2-oxo-4-methoxy-pyrimidin-1-yl, 2-oxo-4-mercapto-pyrimidin-1-yl,    2-oxo-4-hydroxy-pyrimidin-1-yl (i.e., uracinyl),    2-oxo-4-hydroxy-5-methylpyrimidin-1-yl (i.e., thyminyl),    4-amino-5-methyl-2-oxo-pyrimidin-1-yl (i.e., 5-methylcytosinyl)    group or a 4-amino-5-methyl-2-oxo-pyrimidin-1-yl group the amino    group of which is protected, and pharmacologically acceptable salts    thereof; and-   (10) oligonucleotide analogues in which B is a    6-benzoylaminopurin-9-yl, adeninyl,    2-isobutyrylamino-6-hydroxypurin-9-yl, guaninyl,    2-oxo-4-benzoylamino-pyrimidin-1-yl, cytosinyl,    2-oxo-5-methyl-4-benzoylamino-pyrimidin-1-yl, 5-methylcytosinyl,    uracinyl or thyminyl group, and pharmacologically acceptable salts    thereof.

The above (9) and (10) indicate the more preferred oligonucleotideanalogues as the number becomes larger, and in the formula (2), theoligonucleotide analogues obtained by optionally selecting A from (8)and optionally selecting B from (9) and (10) or optionally combiningthese and the salts thereof are preferred.

The specific compounds included in the compound of the above formula (1)of the present invention are illustrated in Tables 1 and 2. However, thecompounds of the present invention are not limited to those.

In Table 1 and Table 2, Exe. com. num. represents Exemplificationcompound number, Me represents a methyl group, Bn represents a benzylgroup, Bz represents a benzoyl group, PMB represents a p-methoxybenzylgroup, Tr represents a triphenylmethyl group, MMTr represents a4-methoxytriphenylmethyl (monomethoxytrityl) group, DMTr represents a4,4′-dimethoxytriphenylmethyl (dimethoxytrityl) group, TMTr represents a4,4′,4″-trimethoxytriphenylmethyl (trimethoxytrityl) group, TMSrepresents a trimethylsilyl group, TBDMS represents atert-butyldimethylsilyl group, TBDPS represents atert-butyldiphenylsilyl group and TIPS represents a triisopropylsilylgroup.

TABLE 1 (1′)

Exe. com. num. A R¹ R² R^(3a) R^(4a) 1-1 CH₂ H H H H 1-2 CH₂ H H H NH₂1-3 CH₂ H H H OH 1-4 CH₂ H H OH H 1-5 CH₂ H H OH NH₂ 1-6 CH₂ H H OH OH1-7 CH₂ H H NH₂ H 1-8 CH₂ H H NH₂ NH₂ 1-9 CH₂ H H NH₂ Cl 1-10 CH₂ H HNH₂ F 1-11 CH₂ H H NH₂ Br 1-12 CH₂ H H NH₂ OH 1-13 CH₂ H H OMe H 1-14CH₂ H H OMe OMe 1-15 CH₂ H H OMe NH₂ 1-16 CH₂ H H Cl H 1-17 CH₂ H H Br H1-18 CH₂ H H F H 1-19 CH₂ H H Cl Cl 1-20 CH₂ H H SH H 1-21 CH₂ Bn H NHBzH 1-22 CH₂ Bn H OH NHCOCH(CH₃)₂ 1-23 CH₂ Bn Bn NHBz H 1-24 CH₂ Bn Bn OHNHCOCH(CH₃)₂ 1-25 CH₂ PMB H NHBz H 1-26 CH₂ PMB H OH NHCOCH(CH₃)₂ 1-27CH₂ PMB PMB NHBz H 1-28 CH₂ PMB PMB OH NHCOCH(CH₃)₂ 1-29 CH₂ Tr H NHBz H1-30 CH₂ MMTr H NHBz H 1-31 CH₂ DMTr H NHBz H 1-32 CH₂ TMTr H NHBz H1-33 CH₂ Tr H OH NHCOCH(CH₃)₂ 1-34 CH₂ MMTr H OH NHCOCH(CH₃)₂ 1-35 CH₂DMTr H OH NHCOCH(CH₃)₂ 1-36 CH₂ TMTr H OH NHCOCH(CH₃)₂ 1-37 CH₂ TMS HNHBz H 1-38 CH₂ TBDMS H NHBz H 1-39 CH₂ TBDPS H NHBz H 1-40 CH₂ TIPS HNHBz H 1-41 CH₂ TMS H OH NHCOCH(CH₃)₂ 1-42 CH₂ TBDMS H OH NHCOCH(CH₃)₂1-43 CH₂ TBDPS H OH NHCOCH(CH₃)₂ 1-44 CH₂ TIPS H OH NHCOCH(CH₃)₂ 1-45(CH₂)₂ H H H H 1-46 (CH₂)₂ H H H NH₂ 1-47 (CH₂)₂ H H H OH 1-48 (CH₂)₂ HH OH H 1-49 (CH₂)₂ H H OH NH₂ 1-50 (CH₂)₂ H H OH OH 1-51 (CH₂)₂ H H NH₂H 1-52 (CH₂)₂ H H NH₂ NH₂ 1-53 (CH₂)₂ H H NH₂ Cl 1-54 (CH₂)₂ H H NH₂ F1-55 (CH₂)₂ H H NH₂ Br 1-56 (CH₂)₂ H H NH₂ OH 1-57 (CH₂)₂ H H OMe H 1-58(CH₂)₂ H H OMe OMe 1-59 (CH₂)₂ H H OMe NH₂ 1-60 (CH₂)₂ H H Cl H 1-61(CH₂)₂ H H Br H 1-62 (CH₂)₂ H H F H 1-63 (CH₂)₂ H H Cl Cl 1-64 (CH₂)₂ HH SH H 1-65 (CH₂)₂ Bn H NHBz H 1-66 (CH₂)₂ Bn H OH NHCOCH(CH₃)₂ 1-67(CH₂)₂ Bn Bn NHBz H 1-68 (CH₂)₂ Bn Bn OH NHCOCH(CH₃)₂ 1-69 (CH₂)₂ PMB HNHBz H 1-70 (CH₂)₂ PMB H OH NHCOCH(CH₃)₂ 1-71 (CH₂)₂ PMB PMB NHBz H 1-72(CH₂)₂ PMB PMB OH NHCOCH(CH₃)₂ 1-73 (CH₂)₂ Tr H NHBz H 1-74 (CH₂)₂ MMTrH NHBz H 1-75 (CH₂)₂ DMTr H NHBz H 1-76 (CH₂)₂ TMTr H NHBz H 1-77 (CH₂)₂Tr H OH NHCOCH(CH₃)₂ 1-78 (CH₂)₂ MMTr H OH NHCOCH(CH₃)₂ 1-79 (CH₂)₂ DMTrH OH NHCOCH(CH₃)₂ 1-80 (CH₂)₂ TMTr H OH NHCOCH(CH₃)₂ 1-81 (CH₂)₂ TMS HNHBz H 1-82 (CH₂)₂ TBDMS H NHBz H 1-83 (CH₂)₂ TBDPS H NHBz H 1-84 (CH₂)₂TIPS H NHBz H 1-85 (CH₂)₂ TMS H OH NHCOCH(CH₃)₂ 1-86 (CH₂)₂ TBDMS H OHNHCOCH(CH₃)₂ 1-87 (CH₂)₂ TBDPS H OH NHCOCH(CH₃)₂ 1-88 (CH₂)₂ TIPS H OHNHCOCH(CH₃)₂ 1-89 (CH₂)₃ H H H H 1-90 (CH₂)₃ H H H NH₂ 1-91 (CH₂)₃ H H HOH 1-92 (CH₂)₃ H H OH H 1-93 (CH₂)₃ H H OH NH₂ 1-94 (CH₂)₃ H H OH OH1-95 (CH₂)₃ H H NH₂ H 1-96 (CH₂)₃ H H NH₂ NH₂ 1-97 (CH₂)₃ H H NH₂ Cl1-98 (CH₂)₃ H H NH₂ F 1-99 (CH₂)₃ H H NH₂ Br 1-100 (CH₂)₃ H H NH₂ OH1-101 (CH₂)₃ H H OMe H 1-102 (CH₂)₃ H H OMe OMe 1-103 (CH₂)₃ H H OMe NH₂1-104 (CH₂)₃ H H Cl H 1-105 (CH₂)₃ H H Br H 1-106 (CH₂)₃ H H F H 1-107(CH₂)₃ H H Cl Cl 1-108 (CH₂)₃ H H SH H 1-109 (CH₂)₃ Bn H NHBz H 1-110(CH₂)₃ Bn H OH NHCOCH(CH₃)₂ 1-111 (CH₂)₃ Bn Bn NHBz H 1-112 (CH₂)₃ Bn BnOH NHCOCH(CH₃)₂ 1-113 (CH₂)₃ PMB H NHBz H 1-114 (CH₂)₃ PMB H OHNHCOCH(CH₃)₂ 1-115 (CH₂)₃ PMB PMB NHBz H 1-116 (CH₂)₃ PMB PMB OHNHCOCH(CH₃)₂ 1-117 (CH₂)₃ Tr H NHBz H 1-118 (CH₂)₃ MMTr H NHBz H 1-119(CH₂)₃ DMTr H NHBz H 1-120 (CH₂)₃ TMTr H NHBz H 1-121 (CH₂)₃ Tr H OHNHCOCH(CH₃)₂ 1-122 (CH₂)₃ MMTr H OH NHCOCH(CH₃)₂ 1-123 (CH₂)₃ DMTr H OHNHCOCH(CH₃)₂ 1-124 (CH₂)₃ TMTr H OH NHCOCH(CH₃)₂ 1-125 (CH₂)₃ TMS H NHBzH 1-126 (CH₂)₃ TBDMS H NHBz H 1-127 (CH₂)₃ TBDPS H NHBz H 1-128 (CH₂)₃TIPS H NHBz H 1-129 (CH₂)₃ TMS H OH NHCOCH(CH₃)₂ 1-130 (CH₂)₃ TBDMS H OHNHCOCH(CH₃)₂ 1-131 (CH₂)₃ TBDPS H OH NHCOCH(CH₃)₂ 1-132 (CH₂)₃ TIPS H OHNHCOCH(CH₃)₂ 1-133 (CH₂)₄ H H H H 1-134 (CH₂)₄ H H H NH₂ 1-135 (CH₂)₄ HH H OH 1-136 (CH₂)₄ H H OH H 1-137 (CH₂)₄ H H OH NH₂ 1-138 (CH₂)₄ H H OHOH 1-139 (CH₂)₄ H H NH₂ H 1-140 (CH₂)₄ H H NH₂ NH₂ 1-141 (CH₂)₄ H H NH₂Cl 1-142 (CH₂)₄ H H NH₂ F 1-143 (CH₂)₄ H H NH₂ Br 1-144 (CH₂)₄ H H NH₂OH 1-145 (CH₂)₄ H H OMe H 1-146 (CH₂)₄ H H OMe OMe 1-147 (CH₂)₄ H H OMeNH₂ 1-148 (CH₂)₄ H H Cl H 1-149 (CH₂)₄ H H Br H 1-150 (CH₂)₄ H H F H1-151 (CH₂)₄ H H Cl Cl 1-152 (CH₂)₄ H H SH H 1-153 (CH₂)₄ Bn H NHBz H1-154 (CH₂)₄ Bn H OH NHCOCH(CH₃)₂ 1-155 (CH₂)₄ Bn Bn NHBz H 1-156 (CH₂)₄Bn Bn OH NHCOCH(CH₃)₂ 1-157 (CH₂)₄ PMB H NHBz H 1-158 (CH₂)₄ PMB H OHNHCOCH(CH₃)₂ 1-159 (CH₂)₄ PMB PMB NHBz H 1-160 (CH₂)₄ PMB PMB OHNHCOCH(CH₃)₂ 1-161 (CH₂)₄ Tr H NHBz H 1-162 (CH₂)₄ MMTr H NHBz H 1-163(CH₂)₄ DMTr H NHBz H 1-164 (CH₂)₄ TMTr H NHBz H 1-165 (CH₂)₄ Tr H OHNHCOCH(CH₃)₂ 1-166 (CH₂)₄ MMTr H OH NHCOCH(CH₃)₂ 1-167 (CH₂)₄ DMTr H OHNHCOCH(CH₃)₂ 1-168 (CH₂)₄ TMTr H OH NHCOCH(CH₃)₂ 1-169 (CH₂)₄ TMS H NHBzH 1-170 (CH₂)₄ TBDMS H NHBz H 1-171 (CH₂)₄ TBDPS H NHBz H 1-172 (CH₂)₄TIPS H NHBz H 1-173 (CH₂)₄ TMS H OH NHCOCH(CH₃)₂ 1-174 (CH₂)₄ TBDMS H OHNHCOCH(CH₃)₂ 1-175 (CH₂)₄ TBDPS H OH NHCOCH(CH₃)₂ 1-176 (CH₂)₄ TIPS H OHNHCOCH(CH₃)₂ 1-177 CH₂ H H OH NHCOCH(CH₃)₂ 1-178 CH₂ H H NHBz H 1-179(CH₂)₂ H H OH NHCOCH(CH₃)₂ 1-180 (CH₂)₂ H H NHBz H 1-181 (CH₂)₃ H H OHNHCOCH(CH₃)₂ 1-182 (CH₂)₃ H H NHBz H 1-183 (CH₂)₄ H H OH NHCOCH(CH₃)₂1-184 (CH₂)₄ H H NHBz H 1-185 CH₂ DMTr P(N(iPr)₂)(OC₂H₄CN) OHNHCOCH(CH₃)₂ 1-186 CH₂ DMTr P(N(iPr)₂)(OC₂H₄CN) NHBz H 1-187 (CH₂)₂ DMTrP(N(iPr)₂)(OC₂H₄CN) OH NHCOCH(CH₃)₂ 1-188 (CH₂)₂ DMTrP(N(iPr)₂)(OC₂H₄CN) NHBz H 1-189 (CH₂)₃ DMTr P(N(iPr)₂)(OC₂H₄CN) OHNHCOCH(CH₃)₂ 1-190 (CH₂)₃ DMTr P(N(iPr)₂)(OC₂H₄CN) NHBz H 1-191 (CH₂)₄DMTr P(N(iPr)₂)(OC₂H₄CN) OH NHCOCH(CH₃)₂ 1-192 (CH₂)₄ DMTrP(N(iPr)₂)(OC₂H₄CN) NHBz H 1-193 CH₂ DMTr P(N(iPr)₂)(OCH₃) OHNHCOCH(CH₃)₂ 1-194 CH₂ DMTr P(N(iPr)₂)(OCH₃) NHBz H 1-195 (CH₂)₂ DMTrP(N(iPr)₂)(OCH₃) OH NHCOCH(CH₃)₂ 1-196 (CH₂)₂ DMTr P(N(iPr)₂)(OCH₃) NHBzH 1-197 (CH₂)₃ DMTr P(N(iPr)₂)(OCH₃) OH NHCOCH(CH₃)₂ 1-198 (CH₂)₃ DMTrP(N(iPr)₂)(OCH₃) NHBz H 1-199 (CH₂)₄ DMTr P(N(iPr)₂)(OCH₃) OHNHCOCH(CH₃)₂ 1-200 (CH₂)₄ DMTr P(N(iPr)₂)(OCH₃) NHBz H 1-201 CH₂ DMTrP(O)(OH)H OH NHCOCH(CH₃)₂ 1-202 CH₂ DMTr P(O)(OH)H NHBz H 1-203 (CH₂)₂DMTr P(O)(OH)H OH NHCOCH(CH₃)₂ 1-204 (CH₂)₂ DMTr P(O)(OH)H NHBz H 1-205(CH₂)₃ DMTr P(O)(OH)H OH NHCOCH(CH₃)₂ 1-206 (CH₂)₃ DMTr P(O)(OH)H NHBz H1-207 (CH₂)₄ DMTr P(O)(OH)H OH NHCOCH(CH₃)₂ 1-208 (CH₂)₄ DMTr P(O)(OH)HNHBz H

TABLE 2 (1″)

Exe. com. num. A R¹ R² R⁵ R⁶ 2-1 CH₂ H H OH H 2-2 CH₂ H H OH CH₃ 2-3 CH₂H H NH₂ H 2-4 CH₂ H H NH₂ CH₃ 2-5 CH₂ H H NH₂ F 2-6 CH₂ H H Cl H 2-7 CH₂H H OMe H 2-8 CH₂ H H SH H 2-9 CH₂ Bn H OH H 2-10 CH₂ Bn Bn OH H 2-11CH₂ PMB H OH H 2-12 CH₂ PMB PMB OH H 2-13 CH₂ Tr H OH H 2-14 CH₂ MMTr HOH H 2-15 CH₂ DMTr H OH H 2-16 CH₂ TMTr H OH H 2-17 CH₂ TMS H OH H 2-18CH₂ TBDMS H OH H 2-19 CH₂ TBDPS H OH H 2-20 CH₂ TIPS H OH H 2-21 CH₂ BnH OH CH₃ 2-22 CH₂ Bn Bn OH CH₃ 2-23 CH₂ PMB H OH CH₃ 2-24 CH₂ PMB PMB OHCH₃ 2-25 CH₂ Tr H OH CH₃ 2-26 CH₂ MMTr H OH CH₃ 2-27 CH₂ DMTr H OH CH₃2-28 CH₂ TMTr H OH CH₃ 2-29 CH₂ TMS H OH CH₃ 2-30 CH₂ TBDMS H OH CH₃2-31 CH₂ TBDPS H OH CH₃ 2-32 CH₂ TIPS H OH CH₃ 2-33 CH₂ Bn H NHBz H 2-34CH₂ Bn Bn NHBz H 2-35 CH₂ PMB H NHBz H 2-36 CH₂ PMB PMB NHBz H 2-37 CH₂Tr H NHBz H 2-38 CH₂ MMTr H NHBz H 2-39 CH₂ DMTr H NHBz H 2-40 CH₂ TMTrH NHBz H 2-41 CH₂ TMS H NHBz H 2-42 CH₂ TBDMS H NHBz H 2-43 CH₂ TBDPS HNHBz H 2-44 CH₂ TIPS H NHBz H 2-45 CH₂ Bn H NHBz CH₃ 2-46 CH₂ Bn Bn NHBzCH₃ 2-47 CH₂ PMB H NHBz CH₃ 2-48 CH₂ PMB PMB NHBz CH₃ 2-49 CH₂ Tr H NHBzCH₃ 2-50 CH₂ MMTr H NHBz CH₃ 2-51 CH₂ DMTr H NHBz CH₃ 2-52 CH₂ TMTr HNHBz CH₃ 2-53 CH₂ TMS H NHBz CH₃ 2-54 CH₂ TBDMS H NHBz CH₃ 2-55 CH₂TBDPS H NHBz CH₃ 2-56 CH₂ TIPS H NHBz CH₃ 2-57 (CH₂)₂ H H OH H 2-58(CH₂)₂ H H OH CH₃ 2-59 (CH₂)₂ H H NH₂ H 2-60 (CH₂)₂ H H NH₂ CH₃ 2-61(CH₂)₂ H H NH₂ F 2-62 (CH₂)₂ H H Cl H 2-63 (CH₂)₂ H H OMe H 2-64 (CH₂)₂H H SH H 2-65 (CH₂)₂ Bn H OH H 2-66 (CH₂)₂ Bn Bn OH H 2-67 (CH₂)₂ PMB HOH H 2-68 (CH₂)₂ PMB PMB OH H 2-69 (CH₂)₂ Tr H OH H 2-70 (CH₂)₂ MMTr HOH H 2-71 (CH₂)₂ DMTr H OH H 2-72 (CH₂)₂ TMTr H OH H 2-73 (CH₂)₂ TMS HOH H 2-74 (CH₂)₂ TBDMS H OH H 2-75 (CH₂)₂ TBDPS H OH H 2-76 (CH₂)₂ TIPSH OH H 2-77 (CH₂)₂ Bn H OH CH₃ 2-78 (CH₂)₂ Bn Bn OH CH₃ 2-79 (CH₂)₂ PMBH OH CH₃ 2-80 (CH₂)₂ PMB PMB OH CH₃ 2-81 (CH₂)₂ Tr H OH CH₃ 2-82 (CH₂)₂MMTr H OH CH₃ 2-83 (CH₂)₂ DMTr H OH CH₃ 2-84 (CH₂)₂ TMTr H OH CH₃ 2-85(CH₂)₂ TMS H OH CH₃ 2-86 (CH₂)₂ TBDMS H OH CH₃ 2-87 (CH₂)₂ TBDPS H OHCH₃ 2-88 (CH₂)₂ TIPS H OH CH₃ 2-89 (CH₂)₂ Bn H NHBz H 2-90 (CH₂)₂ Bn BnNHBz H 2-91 (CH₂)₂ PMB H NHBz H 2-92 (CH₂)₂ PMB PMB NHBz H 2-93 (CH₂)₂Tr H NHBz H 2-94 (CH₂)₂ MMTr H NHBz H 2-95 (CH₂)₂ DMTr H NHBz H 2-96(CH₂)₂ TMTr H NHBz H 2-97 (CH₂)₂ TMS H NHBz H 2-98 (CH₂)₂ TBDMS H NHBz H2-99 (CH₂)₂ TBDPS H NHBz H 2-100 (CH₂)₂ TIPS H NHBz H 2-101 (CH₂)₂ Bn HNHBz CH₃ 2-102 (CH₂)₂ Bn Bn NHBz CH₃ 2-103 (CH₂)₂ PMB H NHBz CH₃ 2-104(CH₂)₂ PMB PMB NHBz CH₃ 2-105 (CH₂)₂ Tr H NHBz CH₃ 2-106 (CH₂)₂ MMTr HNHBz CH₃ 2-107 (CH₂)₂ DMTr H NHBz CH₃ 2-108 (CH₂)₂ TMTr H NHBz CH₃ 2-109(CH₂)₂ TMS H NHBz CH₃ 2-110 (CH₂)₂ TBDMS H NHBz CH₃ 2-111 (CH₂)₂ TBDPS HNHBz CH₃ 2-112 (CH₂)₂ TIPS H NHBz CH₃ 2-113 (CH₂)₃ H H OH H 2-114 (CH₂)₃H H OH CH₃ 2-115 (CH₂)₃ H H NH₂ H 2-116 (CH₂)₃ H H NH₂ CH₃ 2-117 (CH₂)₃H H NH₂ F 2-118 (CH₂)₃ H H Cl H 2-119 (CH₂)₃ H H OMe H 2-120 (CH₂)₃ H HSH H 2-121 (CH₂)₃ Bn H OH H 2-122 (CH₂)₃ Bn Bn OH H 2-123 (CH₂)₃ PMB HOH H 2-124 (CH₂)₃ PMB PMB OH H 2-125 (CH₂)₃ Tr H OH H 2-126 (CH₂)₃ MMTrH OH H 2-127 (CH₂)₃ DMTr H OH H 2-128 (CH₂)₃ TMTr H OH H 2-129 (CH₂)₃TMS H OH H 2-130 (CH₂)₃ TBDMS H OH H 2-131 (CH₂)₃ TBDPS H OH H 2-132(CH₂)₃ TIPS H OH H 2-133 (CH₂)₃ Bn H OH CH₃ 2-134 (CH₂)₃ Bn Bn OH CH₃2-135 (CH₂)₃ PMB H OH CH₃ 2-136 (CH₂)₃ PMB PMB OH CH₃ 2-137 (CH₂)₃ Tr HOH CH₃ 2-138 (CH₂)₃ MMTr H OH CH₃ 2-139 (CH₂)₃ DMTr H OH CH₃ 2-140(CH₂)₃ TMTr H OH CH₃ 2-141 (CH₂)₃ TMS H OH CH₃ 2-142 (CH₂)₃ TBDMS H OHCH₃ 2-143 (CH₂)₃ TBDPS H OH CH₃ 2-144 (CH₂)₃ TIPS H OH CH₃ 2-145 (CH₂)₃Bn H NHBz H 2-146 (CH₂)₃ Bn Bn NHBz H 2-147 (CH₂)₃ PMB H NHBz H 2-148(CH₂)₃ PMB PMB NHBz H 2-149 (CH₂)₃ Tr H NHBz H 2-150 (CH₂)₃ MMTr H NHBzH 2-151 (CH₂)₃ DMTr H NHBz H 2-152 (CH₂)₃ TMTr H NHBz H 2-153 (CH₂)₃ TMSH NHBz H 2-154 (CH₂)₃ TBDMS H NHBz H 2-155 (CH₂)₃ TBDPS H NHBz H 2-156(CH₂)₃ TIPS H NHBz H 2-157 (CH₂)₃ Bn H NHBz CH₃ 2-158 (CH₂)₃ Bn Bn NHBzCH₃ 2-159 (CH₂)₃ PMB H NHBz CH₃ 2-160 (CH₂)₃ PMB PMB NHBz CH₃ 2-161(CH₂)₃ Tr H NHBz CH₃ 2-162 (CH₂)₃ MMTr H NHBz. CH₃ 2-163 (CH₂)₃ DMTr HNHBz CH₃ 2-164 (CH₂)₃ TMTr H NHBz CH₃ 2-165 (CH₂)₃ TMS H NHBz CH₃ 2-166(CH₂)₃ TBDMS H NHBz CH₃ 2-167 (CH₂)₃ TBDPS H NHBz CH₃ 2-168 (CH₂)₃ TIPSH NHBz CH₃ 2-169 (CH₂)₄ H H OH H 2-170 (CH₂)₄ H H OH CH₃ 2-171 (CH₂)₄ HH NH₂ H 2-172 (CH₂)₄ H H NH₂ CH₃ 2-173 (CH₂)₄ H H NH₂ F 2-174 (CH₂)₄ H HCl H 2-175 (CH₂)₄ H H OMe H 2-176 (CH₂)₄ H H SH H 2-177 (CH₂)₄ Bn H OH H2-178 (CH₂)₄ Bn Bn OH H 2-179 (CH₂)₄ PMB H OH H 2-180 (CH₂)₄ PMB PMB OHH 2-181 (CH₂)₄ Tr H OH H 2-182 (CH₂)₄ MMTr H OH H 2-183 (CH₂)₄ DMTr H OHH 2-184 (CH₂)₄ TMTr H OH H 2-185 (CH₂)₄ TMS H OH H 2-186 (CH₂)₄ TBDMS HOH H 2-187 (CH₂)₄ TBDPS H OH H 2-188 (CH₂)₄ TIPS H OH H 2-189 (CH₂)₄ BnH OH CH₃ 2-190 (CH₂)₄ Bn Bn OH CH₃ 2-191 (CH₂)₄ PMB H OH CH₃ 2-192(CH₂)₄ PMB PMB OH CH₃ 2-193 (CH₂)₄ Tr H OH CH₃ 2-194 (CH₂)₄ MMTr H OHCH₃ 2-195 (CH₂)₄ DMTr H OH CH₃ 2-196 (CH₂)₄ TMTr H OH CH₃ 2-197 (CH₂)₄TMS H OH CH₃ 2-198 (CH₂)₄ TBDMS H OH CH₃ 2-199 (CH₂)₄ TBDPS H OH CH₃2-200 (CH₂)₄ TIPS H OH CH₃ 2-201 (CH₂)₄ Bn H NHBz H 2-202 (CH₂)₄ Bn BnNHBz H 2-203 (CH₂)₄ PMB H NHBz H 2-204 (CH₂)₄ PMB PMB NHBz H 2-205(CH₂)₄ Tr H NHBz H 2-206 (CH₂)₄ MMTr H NHBz H 2-207 (CH₂)₄ DMTr H NHBz H2-208 (CH₂)₄ TMTr H NHBz H 2-209 (CH₂)₄ TMS H NHBz H 2-210 (CH₂)₄ TBDMSH NHBz H 2-211 (CH₂)₄ TBDPS H NHBz H 2-212 (CH₂)₄ TIPS H NHBz H 2-213(CH₂)₄ Bn H NHBz CH₃ 2-214 (CH₂)₄ Bn Bn NHBz CH₃ 2-215 (CH₂)₄ PMB H NHBzCH₃ 2-216 (CH₂)₄ PMB PMB NHBz CH₃ 2-217 (CH₂)₄ Tr H NHBz CH₃ 2-218(CH₂)₄ MMTr H NHBz CH₃ 2-219 (CH₂)₄ DMTr H NHBz CH₃ 2-220 (CH₂)₄ TMTr HNHBz CH₃ 2-221 (CH₂)₄ TMS H NHBz CH₃ 2-222 (CH₂)₄ TBDMS H NHBz CH₃ 2-223(CH₂)₄ TBDPS H NHBz CH₃ 2-224 (CH₂)₄ TIPS H NHBz CH₃ 2-225 CH₂ H H NHBzH 2-226 CH₂ H H NHBz CH₃ 2-227 (CH₂)₂ H H NHBz H 2-228 (CH₂)₂ H H NHBzCH₃ 2-229 (CH₂)₃ H H NHBz H 2-230 (CH₂)₃ H H NHBz CH₃ 2-231 (CH₂)₄ H HNHBz H 2-232 (CH₂)₄ H H NHBz CH₃ 2-233 CH₂ DMTr P(N(iPr)₂)(OC₂H₄CN) OH H2-234 CH₂ DMTr P(N(iPr)₂)(OC₂H₄CN) OH CH₃ 2-235 CH₂ DMTrP(N(iPr)₂)(OC₂H₄CN) NHBz H 2-236 CH₂ DMTr P(N(iPr)₂)(OC₂H₄CN) NHBz CH₃2-237 (CH₂)₂ DMTr P(N(iPr)₂)(OC₂H₄CN) OH H 2-238 (CH₂)₂ DMTrP(N(iPr)₂)(OC₂H₄CN) OH CH₃ 2-239 (CH₂)₂ DMTr P(N(iPr)₂)(OC₂H₄CN) NHBz H2-240 (CH₂)₂ DMTr P(N(iPr)₂)(OC₂H₄CN) NHBz CH₃ 2-241 (CH₂)₃ DMTrP(N(iPr)₂)(OC₂H₄CN) OH H 2-242 (CH₂)₃ DMTr P(N(iPr)₂)(OC₂H₄CN) OH CH₃2-243 (CH₂)₃ DMTr P(N(iPr)₂)(OC₂H₄CN) NHBz H 2-244 (CH₂)₃ DMTrP(N(iPr)₂)(OC₂H₄CN) NHBz CH₃ 2-245 (CH₂)₄ DMTr P(N(iPr)₂)(OC₂H₄CN) OH H2-246 (CH₂)₄ DMTr P(N(iPr)₂)(OC₂H₄CN) OH CH₃ 2-247 (CH₂)₄ DMTrP(N(iPr)₂)(OC₂H₄CN) NHBz H 2-248 (CH₂)₄ DMTr P(N(iPr)₂)(OC₂H₄CN) NHBzCH₃ 2-249 CH₂ DMTr P(N(iPr)₂)(OCH₃) OH H 2-250 CH₂ DMTr P(N(iPr)₂)(OCH₃)OH CH₃ 2-251 CH₂ DMTr P(N(iPr)₂)(OCH₃) NHBz H 2-252 CH₂ DMTrP(N(iPr)₂)(OCH₃) NHBz CH₃ 2-253 (CH₂)₂ DMTr P(N(iPr)₂)(OCH₃) OH H 2-254(CH₂)₂ DMTr P(N(iPr)₂)(OCH₃) OH CH₃ 2-255 (CH₂)₂ DMTr P(N(iPr)₂)(OCH₃)NHBz H 2-256 (CH₂)₂ DMTr P(N(iPr)₂)(OCH₃) NHBz CH₃ 2-257 (CH₂)₃ DMTrP(N(iPr)₂)(OCH₃) OH H 2-258 (CH₂)₃ DMTr P(N(iPr)₂)(OCH₃) OH CH₃ 2-259(CH₂)₃ DMTr P(N(iPr)₂)(OCH₃) NHBz H 2-260 (CH₂)₃ DMTr P(N(iPr)₂)(OCH₃)NHBz CH₃ 2-261 (CH₂)₄ DMTr P(N(iPr)₂)(OCH₃) OH H 2-262 (CH₂)₄ DMTrP(N(iPr)₂)(OCH₃) OH CH₃ 2-263 (CH₂)₄ DMTr P(N(iPr)₂)(OCH₃) NHBz H 2-264(CH₂)₄ DMTr P(N(iPr)₂)(OCH₃) NHBz CH₃

In the above Table 1 and Table 2, preferred compounds include thecompounds (1-5), (1-7), (1-23), (1-24), (1-31), (1-35), (1-39), (1-43),(1-49), (1-51), (1-67), (1-68), (1-75), (1-79), (1-83), (1-87), (1-93),(1-95), (1-111), (1-112), (1-119), (1-123), (1-127), (1-131), (1-137),(1-139), (1-155), (1-156), (1-163), (1-167), (1-171), (1-175), (1-177),(1-178), (1-185), (1-186), (1-193), (1-194), (1-201), (1-202), (2-1),(2-2), (2-3), (2-4), (2-10), (2-15), (2-19), (2-22), (2-27), (2-31),(2-34), (2-39), (2-43), (2-46), (2-51), (2-55), (2-57), (2-58), (2-59),(2-60), (2-66), (2-71), (2-75), (2-78), (2-83), (2-87), (2-90), (2-95),(2-99), (2-102), (2-107), (2-111), (2-113), (2-114), (2-115), (2-116),(2-122), (2-127), (2-131), (2-134), (2-139), (2-143), (2-146), (2-151),(2-155), (2-158), (2-163), (2-167), (2-169), (2-170), (2-171), (2-172),(2-178), (2-183), (2-187), (2-190), (2-195), (2-199), (2-202), (2-207),(2-211), (2-214), (2-219), (2-223), (2-225), (2-226), (2-233), (2-234),(2-235) or (2-236), more preferred compounds may include

-   2′-O,4′-C-ethyleneguanosine (1-5),-   2′-O,4′-C-ethyleneadenosine (1-7),-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-6-N-benzoyladenosine (1-23),-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine (1-24),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-6-N-benzoyladenosine (1-31),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine    (1-35),-   2′-O,4′-C-ethylene-2-N-isobutyrylguanosine (1-177),-   2′-O,4′-C-ethylene-6-N-benzoyladenosine (1-178),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite (1-185),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-6-N-benzoyladenosine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite (1-186),-   2′-O,4′-C-ethyleneuridine (2-1),-   2′-O,4′-C-ethylene-5-methyluridine (2-2),-   2′-O,4′-C-ethylenecytidine (2-3),-   2′-O,4′-C-ethylene-5-methylcytidine (2-4),-   3′,5′-di-O-benzyl-2′-O,4′-C-ethyleneuridine (2-10),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethyleneuridine (2-15),-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-5-methyluridine (2-22),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-5-methyluridine (2-27),-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-4-N-benzoylcytidine (2-34),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoylcytidine (2-39),-   3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine    (2-46),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine    (2-51),-   2′-O,4′-C-ethylene-4-N-benzoylcytidine (2-225),-   2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine (2-226),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-uridine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite (2-233),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-5-methyluridine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite (2-234),-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoylcytidine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite (2-235), and-   5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine-3′-O-(2-cyanoethyl    N,N-diisopropyl)phosphoramidite (2-236).

The compound (1) of the present invention can be produced according toProcess A described below.

In Process A, X represents a protecting group; Y represents a protectinggroup; A has the same meaning as defined above; while B¹ represents apurin-9-yl group, a substituted purin-9-yl group or a substituted2-oxo-pyrimidin-1-yl group, said substituents being selected from theabove substituents but with the exclusion of an unprotected amino groupof “an amino group which may be protected”; while B² represents apurin-9-yl group, a substituted purin-9-yl group or a substituted2-oxo-pyrimidin-1-yl group, said substituents being selected from theabove substituents but with the exclusion of protected amino groups of“an amino group which may be protected”; R⁷ represents a group whichforms a leaving group; and R⁸ represents an aliphatic acyl group havingfrom 1 to 4 carbon atoms.

The protecting group of X is the same group as “the hydroxyl protectinggroup” in the above R¹.

The protecting group of Y is the same group as “the hydroxyl protectinggroup” in the above R².

“The group which forms a leaving group” of R⁷ may include a loweralkylsulfonyl group such as methanesulfonyl and ethanesulfonyl; ahalogen-substituted lower alkylsulfonyl group such astrifluoromethanesulfonyl; and an arylsulfonyl group such asp-toluenesulfonyl; preferably a methanesulfonyl group or ap-toluenesulfonyl group.

“The aliphatic acyl group having from 2 to 4 carbon atoms” of R⁸ mayinclude acetyl, propionyl, butyryl groups and the like, preferably anacetyl group.

In the following, each step of Process A will be described in detail.

(Step A-1)

The present step is to prepare a compound (4) by reacting a compound (3)which can be prepared by Methods B to D described later with a reagentfor introducing a leaving group in the presence of a base catalyst in aninert solvent.

The solvent employable here may include aliphatic hydrocarbons such ashexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons suchas benzene, toluene and xylene; halogenated hydrocarbons such asmethylene chloride, chloroform, carbon tetrachloride, dichloroethane,chlorobenzene and dichlorobenzene; esters such as ethyl formate, ethylacetate, propyl acetate, butyl acetate and diethyl carbonate; etherssuch as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane,dimethoxyethane and diethylene glycol dimethyl ether; ketones such asacetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone andcyclohexanone; nitro compounds such as nitroethane and nitrobenzene;nitriles such as acetonitrile and isobutyronitrile; amides such asformamide, N,N-dimethylformamide, N,N-dimethylacetamide,N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorictriamide; sulfoxides such as sulfolane; and pyridine derivatives;preferably pyridine.

The base catalyst employable here may preferably include a base such astriethylamine, pyridine and dimethylaminopyridine.

The reagent for introducing a leaving group may include alkylsulfonylhalides such as methanesulfonyl chloride and ethanesulfonyl bromide; andarylsulfonyl halides such as p-toluenesulfonyl chloride, preferablymethanesulfonyl chloride and p-toluenesulfonyl chloride.

The reaction temperature varies depending on the starting material, thesolvent, the reagent for introducing a leaving group and the basecatalyst, but is usually from 0° C. to 50° C., preferably from 10° C. to40° C.

The reaction time varies depending on the starting material, thesolvent, the reagent for introducing a leaving group, the base catalystand the reaction temperature, but is usually from 10 minutes to 24hours, preferably from 1 to 10 hours.

After the reaction, the desired compound (4) of the present reaction isobtained, for example, by neutralizing the reaction solution,concentrating the reaction mixture, adding an organic solvent immisciblewith water such as ethyl acetate, washing with water, separating anorganic layer containing the desired compound, drying over anhydrousmagnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization and silica gelcolumn chromatography.

(Step A-2)

The present step is to prepare the compound (5) by reacting the compound(4) prepared in Step A-1 with an acid anhydride in the presence of anacid catalyst in a solvent.

The solvent employable here may include ethers such as diethyl ether,dioxane and tetrahydrofuran; nitriles such as acetonitrile andisobutyronitrile; amides such as formamide, N,N-dimethylformamide,N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone andhexamethylphosphororic triamide; and organic acids such as acetic acid;preferably acetic acid.

The acid catalyst employable here may include inorganic acids such ashydrochloric acid, sulfuric acid and nitric acid, preferably sulfuricacid (particularly concentrated sulfuric acid).

The acid anhydride employable here may include an anhydride of a loweraliphatic carboxylic acid such as acetic anhydride and propionic acidanhydride, preferably acetic anhydride.

The reaction temperature varies depending on the starting material, thesolvent, the acid catalyst and the acid anhydride and is usually from 0°C. to 50° C., preferably from 10° C. to 40° C.

The reaction time varies depending on the starting material, thesolvent, the acid catalyst, the acid anhydride and the reactiontemperature, but is usually from 10 minutes to 12 hours, preferably from30 minutes to 3 hours.

After the reaction, the desired compound (5) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step A-3)

The present step is to prepare the compound (6) by reacting the compound(5) prepared in Step A-2 with a trimethylsilylated compoundcorresponding to the purine or pyrimidine which may have a desiredsubstituent prepared according to a reference (H. Vorbrggen, K.Krolikiewicz and B. Bennua, Chem. Ber., 114, 1234-1255 (1981)) in thepresence of an acid catalyst in an inert solvent.

The solvent employable here may include aromatic hydrocarbons such asbenzene, toluene, xylene; halogenated hydrocarbons such as methylenechloride, chloroform, carbon tetrachloride, 1,2-dichloroethane,chlorobenzene and dichlorobenzene; nitriles such as acetonitrile andisobutyronitrile; amides such as formamide, N,N-dimethylformamide,N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone andhexamethylphosphoric triamide; carbon sulfide; preferably1,2-dichloroethane.

The acid catalyst employable here may include Lewis acid catalysts suchas AlCl₃, SnCl₄, TiCl₄, ZnCl₂, BF₃, trimethylsilyltrifluoromethanesulfonate; preferably trimethylsilyltrifluoromethanesulfonate.

The reaction temperature varies depending on the starting material, thesolvent and the acid catalyst but is usually from 0° C. to 100° C.,preferably from 50° C. to 80° C.

The reaction time varies depending on the starting material, thesolvent, the acid catalyst and the reaction temperature but is usuallyfrom 1 hour to 24 hours, preferably from 1 hour to 8 hours.

After the reaction, the desired compound (6) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step A-4)

The present step is to prepare the compound (1a) of the presentinvention by cyclization of the compound (6) prepared by Step A-3 in thepresence of a base catalyst in an inert solvent.

The solvent employable here may include water; pyridine derivatives;acetonitriles such as acetonitrile and isobutyronitrile; amides such asformamide, N,N-dimethylformamide, N,N-dimethylacetamide,N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorictriamide; and a mixture thereof, preferably a mixture of water andpyridine.

The base catalyst employable here may include alkali metal hydroxidessuch as sodium hydroxide and potassium hydroxide; alkali metalcarbonates such as sodium carbonate and potassium carbonate; alkalimetal alkoxides such as sodium methoxide and sodium ethoxide; andaqueous ammonia; preferably alkali metal hydroxides (particularly sodiumhydroxide).

The reaction temperature varies depending on the starting material, thesolvent and the base catalyst but is usually from 0° C. to 50° C.,preferably from 10° C. to 30° C.

The reaction time varies depending on the starting material, thesolvent, the acid catalyst and the reaction temperature but is usuallyfrom 1 minute to 5 hours, preferably from 1 minute to 30 minutes.

After the reaction, the desired compound (1a) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step A-5)

The present step is to prepare the compound (1b) by reacting thecompound (1a) obtained by Step A-4 with a deprotecting reagent in aninert solvent.

The deprotection method varies depending on the kind of protecting groupand is not particularly limited unless it causes other side reactionsand can be carried out, for example, by a method described in“Protective Groups in Organic Synthesis” (Theodora W. Greene and PeterG. M. Wuts, 1999, Published by A Wiley-Interscience Publication).

Particularly, the deprotection method can be carried out by thefollowing methods in the case where the protecting group is (1) “analiphatic acyl group or an aromatic acyl group”, (2) “a methyl groupsubstituted by from 1 to 3 aryl groups” or “a methyl group substitutedby from 1 to 3 aryl groups the aryl ring of which is substituted bylower alkyl, lower alkoxy, halogen or cyano group” or (3) “a silylgroup”.

(1) In the case where the protecting group is an aliphatic acyl group oran aromatic acyl group, the deprotection reaction is usually carried outby treating it with a base in an inert solvent.

The solvent employable here is not particularly limited so long as it iseasily mixed with water, does not inhibit the reaction and dissolves thestarting material to some extent and may include aqueous or anhydrousamides such as dimethylformamide and dimethylacetamide; halogenatedhydrocarbons such as methylene chloride, chloroform, 1,2-dichloroethaneor carbon tetrachloride; and ethers such as tetrahydrofuran, diethylether and dioxane; preferably ethers, more preferably tetrahydrofuran.

The base employable here may include alkali metal hydroxides such aslithium hydroxide, potassium hydroxide and sodium hydroxide; alkalimetal carbonates such as sodium carbonate and potassium carbonate;alkali metal alkoxides such as sodium methoxide and sodium ethoxide; andan ammonia solution such as aqueous ammonia and ammonia/methanolsolution.

The reaction temperature is from 0° C. to 60° C., preferably from 20° C.to 40° C.

The reaction time is from 10 minutes to 24 hours, preferably from 1 hourto 3 hours.

After the reaction, the desired compound (1b) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(2) In the case where the protecting group is “a methyl groupsubstituted by from one to three aryl groups” or “a methyl groupsubstituted by from one to three aryl groups the aryl ring of which issubstituted by a lower alkyl, lower alkoxy, halogen or cyano group”, thereaction is carried out in an inert solvent using a reducing agent.

The solvent employable here may preferably include alcohols such asmethanol, ethanol and isopropanol; ethers such as diethyl ether,tetrahydrofuran and dioxane; aromatic hydrocarbons such as toluene,benzene and xylene; aliphatic hydrocarbons such as hexane andcyclohexane; esters such as ethyl acetate and propyl acetate; organicacids such as acetic acid; or a mixture of these organic solvents andwater.

The reducing agent employable here is not particularly limited so longas it is usually used for a catalytic reduction and may preferablyinclude palladium on carbon, Raney nickel, platinum oxide, platinumblack, rhodium-aluminum oxide, triphenylphosphine-rhodium chloride andpalladium-barium sulfate.

The pressure is not particularly limited but is usually from 1 to 10atm.

The reaction temperature is from 0° C. to 60° C., preferably from 20° C.to 40° C.

The reaction time is from 10 minutes to 24 hours, preferably from onehour to three hours.

After the reaction, the desired compound (1b) of the present reaction isobtained, for example, by removing the reducing agent from the reactionmixture, adding an organic solvent immiscible with water such as ethylacetate, washing with water, separating an organic layer containing thedesired compound, drying over anhydrous magnesium sulfate and distillingoff the solvent. The desired product thus obtained can be furtherpurified, if necessary, by a conventional method, for example,recrystallization, silica gel column chromatography and the like.

In the case where the protecting group is “a methyl group substituted bythree aryl groups”, i.e., a trityl group, the deprotection reaction canbe also carried out using an acid.

In this case, the solvent employable here may include aromatichydrocarbons such as benzene, toluene and xylene; halogenatedhydrocarbons such as methylene chloride, chloroform, carbontetrachloride, 1,2-dichloroethane, chlorobenzene and dichlorobenzene;alcohols such as methanol, ethanol, isopropanol and tert-butanol;nitriles such as acetonitrile and isobutyronitrile; amides such asformamide, N,N-dimethylformamide, N,N-dimethylacetamide,N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorictriamide; and organic acids such as acetic acid; preferably organicacids (particularly acetic acid) or alcohols (particularlytert-butanol):

The acid employable here may preferably include acetic acid ortrifluoroacetic acid.

The reaction temperature is from 0° C. to 60° C., preferably from 20° C.to 40° C.

The reaction time is from 10 minutes to 24 hours, preferably from one 1to 3 hours.

After the reaction, the desired compound (1b) of the present reaction isobtained, for example, by neutralizing the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(3) In the case where the protecting group is “a silyl group”, it canusually be removed by treating with a compound producing a fluorineanion such as tetrabutylammonium fluoride, hydrofluoric acid,hydrofluoric acid-pyridine and potassium fluoride, or organic acids suchas acetic acid, methanesulfonic acid, para-toluenesulfonic acid,trifluoroacetic acid and trifluoromethanesulfonic acid, or inorganicacids such as hydrochloric acid.

In the case where the protecting group is removed by a fluorine anion,the reaction is sometimes promoted by adding organic acids such asformic acid, acetic acid and propionic acid thereto.

The solvent employable here is not particularly limited so long as itdoes not inhibit the reaction and dissolves the starting material tosome extent and may preferably include ethers such as diethyl ether,diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane anddiethylene glycol dimethyl ether; nitriles such as acetonitrile andisobutyronitrile; water; organic acids such as acetic acid; and amixture thereof.

The reaction temperature is from 0° C. to 100° C., preferably from 20°C. to 70° C.

The reaction time is from 5 minutes to 48 hours, preferably from onehour to 24 hours.

After the reaction, the desired compound (1b) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step A-6)

The present step is to prepare the compound (1c) of the presentinvention by reacting the compound (1b) obtained in Step A-5 with adeprotection reagent in an inert solvent.

The deprotection method varies depending on the kind of protecting groupand is not particularly limited so long as it does not cause other sidereactions and can be carried out, for example, by a method described in“Protective Groups in Organic Synthesis” (by Theodora W. Greene, 1981,published by A Wiley-Interscience Publication).

Particularly, the deprotection method can be carried out by thefollowing method in the case where the protecting group is an aliphaticacyl group or an aromatic acyl group.

Namely, the deprotection method is usually carried out by reacting witha base in an inert solvent in the case where the protecting group is analiphatic acyl group or an aromatic acyl group.

The solvent employable here is not particularly limited so long as it iseasily mixed with water, does not inhibit the reaction and dissolves thestarting material to some extent and may include aqueous or anhydrousalcohols such as methanol and ethanol; amides such as dimethylformamideand dimethylacetamide; halogenated hydrocarbons such as methylenechloride, chloroform, 1,2-dichloroethane or carbon tetrachloride; andethers such as tetrahydrofuran, diethyl ether and dioxane; preferablyalcohols; more preferably methanol.

The base employable here may include alkali metal hydroxides such aslithium hydroxide, potassium hydroxide and sodium hydroxide; alkalimetal carbonates such as sodium carbonate and potassium carbonate;alkali metal alkoxides such as sodium methoxide and sodium ethoxide; andammonia; preferably ammonia.

The reaction temperature is from 0° C. to 50° C., preferably from 10° C.to 40° C.

The reaction time is from 10 minutes to 24 hours, preferably from 10minutes to 15 hours. After the reaction, the desired compound (1c) ofthe present reaction is obtained, for example, by concentrating thereaction mixture, adding an organic solvent immiscible with water suchas ethyl acetate, washing with water, separating an organic layercontaining the desired compound, drying over anhydrous magnesium sulfateand distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

The intermediate (3) described above can be prepared by Processes B to Ddescribed below.

In Processes B to D, X and Y have the same meanings as defined above; R⁹represents a group which forms a leaving group; E represents anethylene, trimethylene or tetramethylene group; and Z represents asingle bond, a methylene or ethylene group.

The group which forms a leaving group of R⁹ may include the groupdescribed in the above R⁷, preferably a trifluoromethanesulfonyl group.

R¹¹ and R¹² are the same and represent a hydrogen atom or taken togetherform an oxygen atom.

In the case where R¹¹ and R¹² taken together form the oxygen atom, R¹⁰represents an alkyl group having from 1 to 4 carbon atoms such asmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl andtert-butyl, preferably a methyl group. In the case where R¹¹ and R¹² arethe same and represent a hydrogen atom, R¹⁰ may include an aralkyl groupsuch as a benzyl group; an alkoxyalkyl group such as a methoxymethylgroup; an arylcarbonyloxymethyl group such as a benzoyloxymethyl group,an aralkyloxymethyl group such as a benzyloxymethyl group; analkoxyalkoxyalkyl group such as a methoxyethoxymethyl group; a silylgroup such as trimethylsilyl, t-butyldimethylsilyl, diphenylmethylsilyl,diphenylbutylsilyl, diphenylisopropylsilyl and phenyldiisopropylsilyl.

The compound (7), i.e., the starting material used in Process B orProcess C can be prepared by the following method.

Namely, a compound corresponding to the compound (6) of which the “X”moiety is a hydrogen atom is prepared from 1,1,5,6-diisopropylideneD-glucose on public sale according to the method of the literature (R.D. Youssefyeh, J. P. H. Verheyden, J. G. Moffatt. J. Org. Chem., 44,1301-1309 (1979)) and subsequently the compound (6) can be preparedaccording to the method of the literature (T. Waga, T. Nishizaki, I.Miyakawa, H. Ohrui, H. Meguro, Biosci. Biotechnol. Biochem., 57,1433-1438 (1993)) (in the case of X═Bn).

(Process B)

(Step B-1)

The present step is to prepare the compound (8) by reacting the compound(7) prepared by the above method with a reagent for introducing aleaving group in the presence of a base catalyst in an inert solvent.

The solvent employable here may include amides such as dimethylformamideand dimethylacetamide; halogenated hydrocarbons such as methylenechloride, chloroform, 1,2-dichloroethane or carbon tetrachloride; andethers such as tetrahydrofuran, diethyl ether and dioxane; preferablymethylene chloride.

The base catalyst employable here may preferably include a base such astriethylamine, pyridine and dimethylaminopyridine.

The reagent employable for introducing a leaving group may preferablyinclude trifluoromethanesulfonic acid chloride ortrifluoromethanesulfonic anhydride.

The reaction temperature varies depending on the starting material, thesolvent and the acid catalyst, but is usually from −100° C. to −50° C.,preferably from −100° C. to −70° C.

The reaction time varies depending on the starting material, thesolvent, the acid catalyst and the reaction temperature but is usuallyfrom 30 minutes to 12 hours, preferably from 30 minutes to 3 hours.

After the reaction, the desired compound (8) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step B-2)

The present step is to prepare the compound (9) by reacting the compound(8) prepared by Step B-1 with a cyanating reagent in an inert solvent.

The solvent employable here may include amides such as dimethylformamideand dimethylacetamide; halogenated hydrocarbons such as methylenechloride, chloroform, 1,2-dichloroethane or carbon tetrachloride; etherssuch as tetrahydrofuran, diethyl ether and dioxane; acetonitrile;dimethylsulfoxide and the like; preferably amides (dimethylformamide).

The cyanating reagent employable here may include KCN, NaCN andtrimethylsilane cyanide, preferably NaCN.

The reaction temperature varies depending on the starting material, thesolvent and the cyanating reagent but is usually from 0° C. to 100° C.,preferably from 30° C. to 70° C.

The reaction time varies depending on the starting material, thesolvent, the cyanating reagent and the reaction temperature but isusually from 30 minutes to 12 hours, preferably from one 1 to 3 hours.

After the reaction, the desired compound (9) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step B-3)

The present step is to prepare the compound (10) by reacting thecompound (9) prepared in Step B-2 with a reducing agent in an inertsolvent.

The solvent employable here may include halogenated hydrocarbons such asmethylene chloride, chloroform, 1,2-dichloroethane or carbontetrachloride; aliphatic hydrocarbons such as hexane, heptane, ligroinand petroleum ether; aromatic hydrocarbons such as benzene, toluene andxylene; ethers such as diethyl ether, diisopropyl ether,tetrahydrofuran, dioxane, dimethoxyethane and diethylene glycol dimethylether; and ketones such as acetone, methyl ethyl ketone, methyl isobutylketone isophorone and cyclohexanone; preferably halogenated hydrocarbons(particularly methylene chloride).

The reducing agent employable here may include diisobutyl aluminumhydride and triethoxy aluminum hydride, preferably diisobutyl aluminumhydride.

The reaction temperature varies depending on the starting material, thesolvent and the reducing agent but is usually from −100° C. to −50° C.,preferably from −90° C. to −70° C.

The reaction time varies depending on the starting material, thesolvent, the reducing agent and the reaction temperature but is usuallyfrom 30 minutes to 12 hours, preferably from 1 hour to 5 hours.

After the reaction, the desired compound (10) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step B-4)

The present step is to prepare the compound (3a), one of the startingmaterials of Process A by reacting the compound (10) prepared in StepB-3 with a reducing agent in an inert solvent.

The solvent employable here may include alcohols such as methanol,ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol,isoamyl alcohol, diethylene glycol, glycerine, octanol, cyclohexanol andmethyl cellosolve; and acetic acid; preferably alcohols (particularlyethanol).

The reducing agent employable here may include alkali metal boronhydrides such as sodium boron hydride and lithium boron hydride;aluminum hydride compounds such as lithium aluminum hydride and lithiumtriethoxide aluminum hydride; and borane; preferably sodium boronhydride.

The reaction temperature varies depending on the starting material, thesolvent and the reducing agent but is usually from 0° C. to 50° C.,preferably from 10° C. to 40° C.

The reaction time varies depending on the starting material, thesolvent, the reducing agent and the reaction temperature but is usuallyfrom 10 minutes to 12 hours, preferably from 30 minutes to 5 hours.

After the reaction, the desired compound (3a) of the present reaction isobtained, for example, by decomposing the reducing agent, concentratingthe reaction mixture, adding an organic solvent immiscible with watersuch as ethyl acetate, washing with water, separating an organic layercontaining the desired compound, drying over anhydrous magnesium sulfateand distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Process C)

(Step C-1)

The present step is to prepare the compound (11) by reacting thecompound (7) prepared in the above process with an oxidizing agent in aninert solvent.

The solvent employable here may include aliphatic hydrocarbons such ashexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons suchas benzene, toluene and xylene; halogenated hydrocarbons such asmethylene chloride, chloroform, carbon tetrachloride, dichloroethane,chlorobenzene and dichlorobenzene; esters such as ethyl formate, ethylacetate, propyl acetate, butyl acetate and diethyl carbonate; etherssuch as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane,dimethoxyethane, diethylene glycol dimethyl ether; and ketones such asacetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone andcyclohexanone; preferably halogenated hydrocarbons (particularlymethylene chloride).

The oxidizing agent employable here may include the Swern reagent foroxidation, the Dess-Martin reagent for oxidation, a chromium trioxidecomplex such as pyridine hydrochloride/chromium trioxide complex(pyridinium chlorochromate and pyridinium dichromate), preferably theSwern reagent for oxidation (namely, dimethyl sulfoxide-oxalylchloride).

The reaction temperature varies depending on the starting material, thesolvent and the oxidizing agent but is usually from −100° C. to −50° C.,preferably from −100° C. to −70° C.

The reaction time varies depending on the starting material, thesolvent, the oxidizing agent and the reaction temperature but is usuallyfrom 30 minutes to 12 hours, preferably from 1 hour to 5 hours.

After the reaction, the desired compound (11) of the present reaction isobtained, for example, by decomposing the oxidizing agent, concentratingthe reaction mixture, adding an organic solvent immiscible with watersuch as ethyl acetate, washing with water, separating an organic layercontaining the desired compound, drying over anhydrous magnesium sulfateand distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step C-2)

The present step is to prepare the compound (12) by reacting thecompound (11) prepared in Step C-1 with a carbon-increasing reagent inan inert solvent.

The solvent employable here may include aliphatic hydrocarbons such ashexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons suchas benzene, toluene and xylene; halogenated hydrocarbons such asmethylene chloride, chloroform, carbon tetrachloride, dichloroethane,chlorobenzene and dichlorobenzene; esters such as ethyl formate, ethylacetate, propyl acetate, butyl acetate and diethyl carbonate; etherssuch as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane,dimethoxyethane, diethylene glycol dimethyl ether, and ketones such asacetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone andcyclohexanone; preferably halogenated hydrocarbons (particularlymethylene chloride).

The reagent employable here may include the Wittig reagent,Horner-Emmons reagent, Peterson reaction reagent, TiCl₄—CH₂Cl₂—Zn systemreaction agent and Tebbe reagent, preferably the Wittig reagent,Horner-Emmons reagent and Tebbe reagent.

The reaction temperature varies depending on the starting material, thesolvent and the carbon-increasing reagent but is usually from −20° C. to20° C., preferably 0° C.

The reaction time varies depending on the starting material, thesolvent, the carbon-increasing reagent and the reaction temperature butis usually from 30 minutes to 12 hours, preferably from 1 hour to 5hours.

After the reaction, the desired compound (12) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step C-3)

The present step is to prepare the compound (3a) by selectivelyintroducing a hydroxyl group to a terminal carbon of olefin of thecompound (12) prepared in Step C-2 in an inert solvent.

The solvent employable here may include aliphatic hydrocarbons such ashexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons suchas benzene, toluene and xylene; halogenated hydrocarbons such asmethylene chloride, chloroform, carbon tetrachloride, dichloroethane,chlorobenzene and dichlorobenzene; esters such as ethyl formate, ethylacetate, propyl acetate, butyl acetate and diethyl carbonate; etherssuch as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane,dimethoxyethane and diethylene glycol dimethyl ether; and ketones suchas acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone andcyclohexanone; preferably ethers (particularly tetrahydrofuran).

The reaction reagent employable here may include borane, disiamylborane, thexyl borane, 9-BBN (9-borabicyclo[3.3.1]nonane), preferablythe 9-BBN.

The reaction temperature varies depending on the starting material, thesolvent and the reagent but is usually from 0° C. to 50° C., preferablyfrom 10° C. to 40° C.

The reaction time varies depending on the starting material, thesolvent, the reagent and the reaction temperature but is usually from 6hours to 48 hours, preferably from 12 hours to 24 hours.

After the reaction, the desired compound (3a) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Process D)

(Step D-1)

The present step is to prepare the compound (13) by reacting thecompound (11) prepared in Step C-1 with a carbon-increasing reagent inan inert solvent.

The solvent employable here may include aliphatic hydrocarbons such ashexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons suchas benzene, toluene and xylene; halogenated hydrocarbons such asmethylene chloride, chloroform, carbon tetrachloride, dichloroethane,chlorobenzene and dichlorobenzene; esters such as ethyl formate, ethylacetate, propyl acetate, butyl acetate and diethyl carbonate; etherssuch as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane,dimethoxyethane and diethylene glycol dimethyl ether, and ketones suchas acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone andcyclohexanone; preferably ethers (particularly tetrahydrofuran), morepreferably halogenated hydrocarbons (particularly methylene chloride).

The carbon-increasing reagent employable here may include the Wittigreagent and Horner-Emmons reagent.

The reaction temperature varies depending on the starting material, thesolvent and the reagent but is usually from −20° C. to 40° C.,preferably from 0° C. to 20° C.

The reaction time varies depending on the starting material, thesolvent, the reagent and the reaction temperature but is usually from 30minutes to 12 hours, preferably from 1 hour to 5 hours.

After the reaction, the desired compound (13) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(Step D-2)

The present step is to prepare the compound (14) by reacting thecompound (13) prepared in Step D-1 with a reducing agent in an inertsolvent.

The present step can be carried out according to (2) of Step A-5. In thecase where R¹⁰ is an optionally substituted benzyl group and R¹¹ and R¹²are hydrogen atoms, the compound (3b) can be directly prepared in thisstep.

(Step D-3)

The present step is to prepare the compound (3b), one of the startingmaterials of Process A by reacting the compound (14) prepared in StepD-2 with a reducing agent.

(a) In the case where R¹¹ and R¹² taken together form an oxygen atom.

The solvent employable here may include alcohols such as methanol,ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol,isoamyl alcohol, diethylene glycol, glycerine, octanol, cyclohexanol andmethyl cellosolve; and acetic acid; preferably alcohols (particularlyethanol).

The reducing agent employable here may include alkali metal boronhydrides such as lithium boron hydride; aluminum hydride compounds suchas lithium aluminum hydride and lithium triethoxide aluminum hydride;and borane; preferably borane and lithium aluminum hydride.

The reaction temperature varies depending on the starting material, thesolvent and the reducing agent but is usually from 0° C. to 50° C.,preferably from 10° C. to 40° C.

The reaction time varies depending on the starting material, thesolvent, the reducing agent and the reaction temperature but is usuallyfrom 10 minutes to 12 hours, preferably from 30 minutes to 5 hours.

After the reaction, the desired compound (3b) of the present reaction isobtained, for example, by decomposing the reducing agent, concentratingthe reaction mixture, adding an organic solvent immiscible with watersuch as ethyl acetate, washing with water, separating an organic layercontaining the desired compound, drying over anhydrous magnesium sulfateand distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

(b) In the case where R¹¹ and R¹² are hydrogen atoms and R¹⁰ is a groupother than a benzyl group.

In the case where R¹⁰ is a silyl group, the present step can be carriedout according to the method of (3) of Step A-5.

In the case where R¹⁰ is an aralkyl group such as a benzyl group; analkoxyalkyl group such as a methoxymethyl group; anarylcarbonyloxymethyl group such as a benzoyloxymethyl group or anaralkyloxymethyl group such as a benzyloxymethyl group; and analkoxyalkoxyalkyl group such as a methoxyethoxymethyl group, an acidcatalyst is used and the acid catalyst used in this case may include anorganic acid such as p-toluenesulfonic acid, trifluoroacetic acid anddichloroacetic acid and a Lewis acid such as BF₃ and AlCl₃.

The solvent employable here may include aromatic hydrocarbons such asbenzene, toluene and xylene; halogenated hydrocarbons such as methylenechloride, chloroform, carbon tetrachloride, 1,2-dichloroethane,chlorobenzene and dichlorobenzene; nitriles such as acetonitrile andisobutyronitrile; amides such as formamide, N,N-dimethylformamide,N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone andhexamethylphosphoric triamide; and carbon sulfide.

The reaction temperature varies depending on the starting material, thesolvent and the acid catalyst but is usually from 0° C. to 50° C.,preferably from 10° C. to 40° C.

The reaction time varies depending on the starting material, thesolvent, the acid catalyst and the reaction temperature and is usuallyfrom 10 minutes to 12 hours, preferably from 30 minutes to 5 hours.

After the reaction, the desired compound (3b) of the present reaction isobtained, for example, by neutralizing the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method, for example, recrystallization, silica gelcolumn chromatography and the like.

Oligonucleotides containing a modified nucleoside or a thioatederivative thereof can be prepared by Process E described below usingthe compound (1) of the present invention.

In Process E, A and B have the same meaning as defined above; R¹³represents a hydroxyl protecting group (particularly a trityl groupwhich may be substituted by a methoxy group); R¹⁴ represents aphosphonyl group or a group formed by reacting mono-substitutedchloro(alkoxy)phosphines or di-substituted alkoxyphosphines describedlater.

(Process E)

(Step E-1)

The present step is to prepare the compound (15) by reacting thecompound (1) prepared in Process A with a protecting reagent in an inertsolvent.

The solvent employable here may preferably include aromatic hydrocarbonssuch as benzene, toluene and xylene; halogenated hydrocarbons such asmethylene chloride, chloroform, carbon tetrachloride, dichloroethane,chlorobenzene and dichlorobenzene; esters such as ethyl formate, ethylacetate, propyl acetate, butyl acetate and diethyl carbonate; etherssuch as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane,dimethoxyethane and diethylene glycol dimethyl ether; ketones such asacetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone andcyclohexanone; nitrated compounds such as nitroethane and nitrobenzene;nitriles such as acetonitrile and isobutyronitrile; amides such asformamide, dimethylformamide (DMF), dimethylacetamide andhexamethylphosphoric triamide; sulfoxides such as dimethyl sulfoxide andsulfolane; aliphatic tertiary amines such as trimethylamine,triethylamine and N-methylmorpholine; and aromatic amines such aspyridine and picoline; more preferably halogenated hydrocarbons(particularly methylene chloride) and aromatic amines (particularlypyridine).

The protecting reagent employable here is not particularly limited solong as only the 5′-position can be selectively protected and it can beremoved under acidic or neutral conditions but may preferably includetriarylmethyl halides such as trityl chloride, monomethoxytritylchloride and dimethoxytrityl chloride.

In the case where triarylmethyl halides are used as the protectingreagent, a base is usually used.

In such case, the base employable here may include heterocyclic aminessuch as pyridine, dimethylaminopyridine and pyrrolidinopyridine; andaliphatic tertiary amines such as trimethylamine and triethylamine;preferably pyridine, dimethylaminopyridine and pyrrolidinopyridine.

In the case where a liquid base is used as the solvent, since the baseitself functions as an acid trapping agent, it is not necessary to addanother base.

The reaction temperature varies depending on the starting material, thereagent and the solvent but is usually from 0° C. to 150° C., preferablyfrom 20° C. to 100° C. The reaction time varies depending on thestarting material, the solvent and the reaction temperature but isusually from 1 hour to 100 hours, preferably from 2 hours to 24 hours.

After the reaction, the desired compound (15) of the present reaction isobtained, for example, by concentrating the reaction mixture, adding anorganic solvent immiscible with water such as ethyl acetate, washingwith water, separating an organic layer containing the desired compound,drying over anhydrous magnesium sulfate and distilling off the solvent.

The desired product thus obtained can be further purified, if necessary,by a conventional method; for example, recrystallization, silica gelcolumn chromatography and the like.

(Step E-2)

The present step is to prepare the compound (16) by reacting thecompound (15) prepared in Step E-1 with mono-substitutedchloro(alkoxy)phosphines or di-substituted alkoxyphosphines usually usedfor amiditation in an inert solvent.

The solvent employable here is not particularly limited so long as itdoes not affect the reaction and may preferably include ethers such astetrahydrofuran, diethyl ether and dioxane; and halogenated hydrocarbonssuch as methylene chloride, chloroform, carbon tetrachloride,dichloroethane, chlorobenzene and dichlorobenzene.

The mono-substituted chloro(alkoxy)phosphines employable here mayinclude phosphine derivatives such aschloro(morpholino)methoxyphosphine,chloro(morpholino)cyanoethoxyphosphine,chloro(dimethylamino)methoxyphosphine,chloro(dimethylamino)cyanoethoxyphosphine,chloro(diisopropylamino)methoxyphosphine andchloro(diisopropylamino)cyanoethoxyphosphine, preferablychloro(morpholino)methoxyphosphine,chloro(morpholino)cyanoethoxyphosphine,chloro(diisopropylamino)methoxyphosphine andchloro(diisopropylamino)cyanoethoxyphosphine.

In the case where the mono-substituted-chloro(alkoxy)phosphines areused, an acid trapping agent is used and in such case, the acid trappingagent employable here may include heterocyclic amines such as pyridineand dimethylaminopyridine; and aliphatic amines such as trimethylamine,triethylamine and diisopropylamine; preferably aliphatic amines(particularly diisopropylamine).

The di-substituted alkoxyphosphines employable here may includephosphine derivatives such as bis(diisopropylamino)cyanoethoxyphosphine,bis(diethylamino)methanesulfonylethoxyphosphine,bis(diisopropylamino)(2,2,2-trichloroethoxy)phosphine andbis(diisopropylamino)(4-chlorophenylmethoxy)phosphine, preferablybis(diisopropylamino)cyanoethoxyphosphine.

In the case where the di-substituted alkoxyphosphines are used, an acidis used, and in such case, the acid employable may preferably includetetrazole, acetic acid or p-toluenesulfonic acid.

The reaction temperature is not particularly limited but is usually from0° C. to 80° C., preferably room temperature.

The reaction time varies depending on the starting material, the reagentand the reaction temperature, but is usually from 5 minutes to 30 hours,preferably from 30 minutes to 10 hours in the case where the reaction iscarried out at room temperature.

After the reaction, the desired compound (16) of the present reaction isobtained, for example, by appropriately neutralizing the reactionmixture, removing insolubles by filtration in the case where they exist,adding an organic solvent immiscible with water such as ethyl acetate,washing with water, separating an organic layer containing the desiredcompound, drying over anhydrous magnesium sulfate and distilling off thesolvent. The desired product thus obtained can be further purified, ifnecessary, by a conventional method, for example, recrystallization,reprecipitation or chromatography and the like.

Alternatively, the present step is to prepare the compound (16) byreacting the compound (15) prepared in Step E-1 withtris-(1,2,4-triazolyl)phosphite in an inert solvent (preferablyhalogenated hydrocarbons such as methylene chloride), followed by theaddition of water to effect H-phosphonation.

The reaction temperature is not particularly limited, but is usuallyfrom −20° C. to 100° C., preferably from 10 to 40° C.

The reaction time varies depending on the starting material, the reagentand the reaction temperature and is usually from 5 minutes to 30 hours,preferably 30 minutes in the case where the reaction is carried out atroom temperature.

After the reaction, the desired compound (16) of the present reaction isobtained, for example, by appropriately neutralizing the reactionmixture, removing insolubles by filtration in the case where they exist,adding an organic solvent immiscible with water such as ethyl acetate,washing with water, separating an organic layer containing the desiredcompound, drying over anhydrous magnesium sulfate and distilling off thesolvent. The desired product thus obtained can be further purified, ifnecessary, by a conventional method, for example, recrystallization,reprecipitation or chromatography and the like.

(Step E-3)

In this step, the target oligonucleotide analogue is produced by anautomated DNA synthesizer using at least one compound (16) prepared instep E-2 and commercially available phosphoramidite reagents requiredfor producing an oligonucleotide analogue of a desired nucleotidesequence in accordance with conventional methods.

An oligonucleotide analogue having a desired nucleotide sequence can besynthesized by a DNA synthesizer such as the Perkin-Elmer Model 392using the phosphoramidite method in accordance with the method describedin the literature (Nucleic Acids Research, 12, 4539 (1984)).

In addition, in the case of converting to a thioate as desired, athioate derivative can be obtained in accordance with the methoddescribed in the literature (Tetrahedron Letters, 32, 3005 (1991), J.Am. Chem. Soc., 112, 1253 (1990)) using, besides sulfur, a reagent thatforms a thioate by reacting with trivalent phosphoric acid such astetraethylthiuram disulfide (TETD, Applied Biosystems Inc.) or Beaucagereagent (Millipore Corp.).

The resulting crude oligonucleotide analogue can be purified by OligoPak(reverse phase chromatocolumn) and the purity of the product can beconfirmed by HPLC analysis.

The chain length of the resulting oligonucleotide analogue is normally 2to 50 units, and preferably 10 to 30 units, in nucleoside units.

The complementary chain formation ability and nuclease enzyme resistanceof the resulting oligonucleotide analogue can be determined according tothe methods described below.

Test Method 1

The hybrid formation ability of the oligonucleotide analogue of thepresent invention with respect to complementary DNA and RNA can bedetermined by annealing the various resulting oligonucleotide analogueswith an oligonucleotide analogue composed of naturally-occurring DNA orRNA having a complementary sequence and measuring the meltingtemperature (Tm value).

A sample solution containing equal amounts of oligonucleotide analogueand naturally-occurring complementary oligonucleotide in sodiumphosphate buffer solution was put into a boiling water bath and thenslowly cooled to room temperature over the course of time (annealing).The temperature of the solution was then raised little by little from20° C. to 90° C. in the cell chamber of a spectrophotometer (e.g.,Shimadzu UV-2100PC) followed by measurement of ultraviolet absorption at260 nm.

Test Method 2 Measurement of Nuclease Enzyme Resistance

To the oligonucleotide in a buffer solution was added a nuclease and themixture was warmed. Examples of nucleases that are used include snakevenom phosphodiesterase, endonuclease P1 and endonuclease S1. Althoughthere are no particular restrictions on the buffer solution provided itis a buffer solution suitable for enzymes, Tris-HCl buffer is used inthe case of snake venom phosphodiesterase, while sodium acetate bufferis used in the case of endonuclease P1. In addition, metal ions areadded to the buffer solution as necessary. Examples of metal ions usedinclude Mg²⁺ in the case of snake venom phosphodiesterase and Zn²⁺ inthe case of endonuclease. The reaction temperature is preferably 0 to100° C., and more preferably 30 to 50° C.

Ethylenediamine tetraacetic acid (EDTA) is added after a predeterminedamount of time followed by heating at 100° C. for 2 minutes in order toquench the reaction.

Examples of methods used to assay the amount of oligonucleotideremaining include a method in which the oligonucleotide is labelled witha radioisotope, etc. followed by assaying the cleavage reaction productwith an image analyzer and so forth, a method in which the cleavagereaction product is assayed by reverse phase high-performance liquidchromatography (HPLC), and a method in which the cleavage reactionproduct is stained with a dye (such as ethidium bromide) and assayed byimage processing using a computer.

Dosage forms of the oligonucleotide analogue having one, or two or morestructures of the formula (2) of the present invention may be tablets,capsules, granules, powders or syrup for oral administration, orinjections or suppositories for parenteral administration. These dosageforms are prepared by well-known methods using carriers such asexcipients (for example, organic excipients such as sugar derivatives,e.g. lactose, sucrose, glucose, mannitol and sorbitol; starchderivatives, e.g. corn starch, potato starch, α-starch and dextrin;cellulose derivatives, e.g. crystalline cellulose; gum arabic; dextran;and Pullulan; and inorganic excipients such as silicate derivatives,e.g. light silicic anhydride, synthesized aluminium silicate, calciumsilicate and magnesium aluminate metasilicate; phosphates, e.g. calciumhydrogenphosphate; carbonates, e.g. calcium carbonate; and sulfates,e.g. calcium sulfate), lubricants (for example, stearic acid; stearicacid metal salts such as calcium stearate and magnesium stearate; talc;colloidal silica; waxes such as bee gum and spermaceti; boric acid;adipic acid; sulfates, e.g. sodium sulfate; glycol; fumaric acid; sodiumbenzoate; DL-leucine; fatty acid sodium salt; laurylsulfates such assodium laurylsulfate and magnesium laurylsulfate; silicic acids such assilicic anhydride and silicic acid hydrate; and the above starchderivatives), binders (for example, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, Macrogol andcompounds similar to the above excipients), disintegrants (for example,cellulose derivatives, such as low-substituted hydroxypropyl cellulose,carboxymethyl cellulose, calcium carboxymethyl cellulose and internallybridged sodium carboxymethyl cellulose; and chemically modifiedstarch-celluloses such as carboxymethyl starch, sodium carboxymethylstarch and bridged polyvinyl pyrrolidone), stabilizers (paraoxybenzoatessuch as methylparaben and propylparaben; alcohols such as chlorobutanol,benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenolderivatives such as phenol and cresol; thimerosal; dehydroacetic acid;and sorbic acid), corrigents (for example, sweeteners, souring agents,flavors, etc. usually used), diluents, etc.

More particularly, pharmaceutical compositions containing the activeingredient of the present invention may be in any form suitable for theintended method of administration. When used for oral use for example,tablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, syrups or elixirsmay be prepared. Compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation. Tablets containing the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipient which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium or sodium carbonate, lactose,calcium or sodium phosphate; granulating and disintegrating agents, suchas maize starch, or alginic acid; binding agents, such as starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc. Tablets may be uncoated or may be coated by knowntechniques including microencapsulation to delay disintegration andadsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxy-benzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oral suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in, admixture with a dispersing or wetting agent,a suspending agent, and one or more preservatives. Suitable dispersingor wetting agents and suspending agents are exemplified by thosedisclosed above. Additional excipients, for example sweetening,flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the present invention may be in theform of a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to known art using those suitable dispersing or wetting agentsand suspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

As noted above, formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in a freeflowing form such as a powder or granules, optionally mixed with abinder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g., sodiumstarch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose) surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropyl methylcellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric coating, to provide release in parts of the gutother than the stomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin, or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

While the dose for a particular patient will vary depending on a varietyof factors including the activity of the specific compound orcomposition employed, the condition of the disease, the age, bodyweight, general health, sex and diet of the patient (e.g., warm bloodedanimals including humans), the time and route of administration,administration methods, the rate of excretion, other drugs which havebeen previously administered to the patient, the severity of thedisease, etc. For example, in the case of oral administration, it isdesirable to administer an active ingredient in an amount of from 0.01mg/kg of body weight (preferably 0.1 mg/kg of body weight) to 1000 mg/kgof body weight (preferably 100 mg/kg of body weight) and in the case ofintravenous administration, it is desirable to administer an activeingredient in an amount of from 0.001 mg/kg of body weight (preferably0.01 mg/kg of body weight) to 100 mg/kg of body weight (preferably 10mg/kg of body weight), as a single dose a day or in divided dose atseveral times for a day respectively.

EXAMPLES Example 13′,5′-di-O-Benzyl-2′-O,4′-C-ethylene-4-N-benzoylcytidine

(Exemplification Compound Number 2-34)

An aqueous 2N sodium hydroxide solution (68 ml) was added to a solutionof the compound obtained in Reference example 11 (6.80 g, 8.86 mmol) inpyridine (136 ml) at 0° C. and the mixture was stirred at roomtemperature for 1 hour. The reaction mixture was neutralized by dropwiseaddition of aqueous 20% acetic acid and extracted with chloroform. Thechloroform layer was washed with saturated aqueous sodium chloridesolution and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (usingdichloromethane:methanol=100:3 as the eluant) to afford the titlecompound (3.33 g, 6.02 mmol, 68%).

¹H-NMR (400 MHz, CDCl₃): 8.64 (2H, brs), 7.89 (2H, d, 7.6 Hz), 7.64-7.60(1H, m), 7.54-7.51 (2H, m), 7.48-7.37 (3H, m), 7.36-7.26 (8H, m), 6.18(1H, s), 4.70 (1H, d, 11 Hz), 4.60 (1H, d, 11 Hz), 4.55 (1H, d, 11 Hz),4.46 (1H, d, 2.9 Hz), 4.42 (1H, d, 11 Hz), 4.10-4.02 (2H, m), 3.89 (1H,d, 2.9 Hz), 3.75 (1H, d, 11 Hz), 3.62 (1H, d, 11 Hz), 2.34-2.26 (1H, m),1.39-1.36 (1H, m).

FAB-MAS (mNBA): 554 (M+H)⁺

Example 2 2′-O,4′-C-ethylene-4-N-benzoylcytidine

(Exemplification Compound Number 2-225)

A solution (31.7 ml) of 1.0 M trichloroborane in dichloromethane wasadded dropwise to a solution of the compound obtained in Example 1 (2.06g, 3.72 mmol) in anhydrous methylenechloride (317 ml) at −78° C. and themixture was stirred at −78° C. for 1 hour. The reaction mixture wasslowly warmed to −20° C. and the reaction vessel was placed into anice-sodium chloride bath and the mixture was stirred at between −20° C.and −10° C. for 2 hours. Methanol (12 ml) was slowly added to themixture and the mixture was stirred for 10 minutes. The pH of thereaction mixture was adjust to 7-8 by dropwise addition of saturatedaqueous sodium hydrogencarbonate solution. The mixture was warmed toroom temperature and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (usingdichloromethane:methanol=100:5 as the eluant) to afford the titlecompound (1.21 g, 3.24 mmol, 87%) as a white solid.

¹H-NMR (500 MHz, DMSO-d₆): 11.23 (1H, brs), 8.70 (1H, d, 7.2 Hz), 8.00(2H, d, 7.5 Hz), 7.3-6 (4H, m), 5.97 (1H, s), 5.35 (1H, dd, 5 and 10Hz), 4.10 (1H, dd, 5 and 10 Hz), 4.03 (1H, d, 3.2 Hz), 3.95-3.85 (2H, m)3.83 (1H, d, 3.2 Hz), 3.65-3.51 (2H, m), 2.06-1.98 (1H, m), 1.26 (1).

FAB-MAS (mNBA): 374 (M+H)⁺

Example 3 2′-O,4′-C-ethylene-cytidine

(Exemplification Compound Number 2-3)

A solution of the compound obtained in Example 2 (0.1 g, 0.268 mmol) inmethanol saturated with ammonia (12 ml) was allowed to stand overnight.The mixture was concentrated to dryness to afford the title compound(0.054 g, 75%) as a white solid.

¹H-NMR (500 MHz, DMSO-d₆): 8.18 (1H, d, 7.4 Hz), 7.10 (2H, br), 5.84(1H, s), 5.69 (1H, d, 7.6 Hz), 5.27-5.24 (2H, m), 3.86 (1H, d, 3.2 Hz),3.90-3.78 (2H, m), 3.76 (1H, d, 3.2 Hz), 3.56 (1H, dd, 5.5 and 12 Hz),3.49 (1H, dd, 5.5 and 12 Hz), 2.01-1.93 (111, dt, 7.5 and 12 Hz), 1.22(1H, dd, 3.6 and 13 Hz).

FAB-MAS (mNBA): 270 (M+H)⁺

Example 4 5′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoylcytidine

(Exemplification Compound Number 2-39)

A solution of the compound obtained in Example 2 (1.29 g, 3.46 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved in anhydrous pyridine (26 ml) under nitrogenatmosphere and 4,4′-dimethoxytritylchloride (1.76 g, 5.18 mmol) wasadded to the solution and the mixture was stirred at room temperatureovernight. A small amount of methanol was added to the reaction mixtureand then the solvent was evaporated in vacuo. The residue waspartitioned between water and chloroform and the organic layer waswashed with saturated aqueous sodium hydrogencarbonate solution andsaturated aqueous sodium chloride solution and concentrated in vacuo.The residue was purified by chromatography on a silica gel column (usingdichloromethane:methanol=100:5 as the eluant) to afford the titlecompound (2.10 g, 3.11 mmol, 90%) as a colorless amorphous solid.

¹H-NMR (270 MHz, DMSO-d₆): 11.27 (1H, brs), 8.59 (1H, m), 6.92-8.01(19H, m), 6.03 (1H, s), 5.56 (1H, m), 4.17 (1H, m), 4.08 (1H, m), 3.86(2H, m), 3.77 (6H, s), 3.24 (2H, m), 1.98 (1H, m), 1.24 (1H, m). FAB-MAS(mNBA): 676 (M+H)⁺

Example 55′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoylcytidine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite

(Exemplification Compound Number 2-235)

A solution of the compound obtained in Example 4 (6.53 g, 9.66 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved under nitrogen atmosphere in anhydrousdichloromethane (142 ml). N,N-diisopropylamine (2.80 ml, 16.1 mmol) wasadded to the solution and then 2-cyanoethylN,N-diisopropylchlorophosphoramidite (2.16 ml, 9.66 mmol) was addeddropwise in an ice bath. The mixture was stirred at room temperature for6 hours. The reaction mixture was washed with saturated aqueous sodiumhydrogencarbonate solution and saturated aqueous sodium chloridesolution and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (usingdichloromethane:triethylamine=50:1−dichloromethane:ethylacetate:triethylamine=60:30:1 as the eluant) to afford the titlecompound (7.10 g, 8.11 mmol, 84%) as a pale white compound.

¹H-NMR (400 MHz, CDCl₃): 1.1-1.2 (12H, m), 1.35 (1H, m), 2.11 (1H, m),2.3 (2H, m), 3.35-3.7 (6H, m), 3.8 (6H, m), 3.9-4.1 (2H, m), 4.33 (1H,m), 4.45 (1H, m), 6.23 (1H, s), 6.9 (4H, m), 7.3-7.9 (15H, m), 8.7-8.8(1H, m).

Example 6 3′,5′-Di-O-benzyl-2′-O,4′-C-ethylene-5-methyluridine

(Exemplification Compound Number 2-22)

An aqueous 2N sodium hydroxide solution (5 ml) and mixture solution (5ml), said mixture solution comprised of pyridine:methanol:water=65:30:5,were added to the compound obtained in Reference example 10 (418 mg,0.62 mmol) in pyridine:methanol:water=65:30:5 (5 ml) at 0° C. and themixture was stirred at room temperature for 15 minutes. The reactionmixture was neutralized with 1N hydrochloric acid and extracted withethyl acetate (about 30 ml). The organic layer was washed with saturatedaqueous sodium hydrogencarbonate solution (about 30 ml) and saturatedaqueous sodium chloride solution (about 30 ml), dried over anhydrousmagnesium sulfate and then concentrated in vacuo. The residue waspurified by chromatography on a silica gel column (using hexane:ethylacetate=1:1 as the eluant) to afford a colorless amorphous solid (228mg, 0.49 mmol, 79%).

¹H-NMR (400 MHz, CDCl₃): 1.35 (1H, d, 13 Hz), 1.41 (3H, s), 2.28 (1H,dt, 9.4 and 13 Hz), 3.60 (1H, d, 11 Hz), 3.76 (1H, d, 11 Hz), 3.94 (1H,d, 3.0 Hz), 4.10 (1H, d, 7.0 Hz), 4.14 (1H, d, 7.0 Hz), 4.31 (1H, d, 3.0Hz), 4.51 (1H, d, 12 Hz), 4.54 (1H, d, 12 Hz), 4.58 (1H, d, 12 Hz), 4.75(1H, d, 12 Hz), 6.06 (1H, s), 7.3 (10H, m), 7.91 (1H, s), 8.42 (1H,brs).

FAB-MAS (mNBA): 465 (M+H)⁺

Example 7 2′-O,4′-C-ethylene-5-methyluridine

(Exemplification Compound Number 2-2)

A solution of the compound obtained in Example 6 (195 mg, 0.42 mmol) inmethanol (10 ml) was stirred under hydrogen atmosphere at atmosphericpressure in the presence of 160 mg of 20% Pd(OH)₂ on carbon as ahydrogenation catalyst for 5 hours. The reaction mixture was filtered inorder to remove catalyst and the filtrate was concentrated in vacuo. Theresidue was purified by chromatography on a silica gel column (usingdichloromethane:methanol=10:1 as the eluant) to afford a colorlesspowder (76 mg, 0.268 mmol, 64%).

¹H-NMR (400 MHz, CD₃OD): 1.33 (1H, dd, 3.8 and 13 Hz), 1.86 (3H, d, 0.9Hz), 1.94 (1H, ddd, 7.5, 11.7 and 13 Hz), 3.68 (1H, d, 12 Hz), 3.75 (1H,d, 12 Hz), 3.9-4.0 (2H, m), 4.05 (1H, d, 3.2 Hz), 4.09 (1H, d, 3.2 Hz),6.00 (1H, s), 8.28 (1H, d, 1.1 Hz).

FAB-MAS (mNBA): 285 (M+H)⁺

Example 8 5′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-5-methyluridine

(Exemplification Compound Number 2-27)

A solution of the compound obtained in Example 7 (1.45 g, 5.10 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved in anhydrous pyridine (44 ml) under nitrogenatmosphere and 4,4′-dimethoxytritylchloride (2.59 g, 7.65 mmol) wasadded to the solution and the mixture was stirred at room temperatureovernight. A small amount of methanol was added to the reaction mixtureand then the solvent was evaporated in vacuo. The residue waspartitioned between water and chloroform and the organic layer waswashed with saturated aqueous sodium hydrogencarbonate solution andsaturated aqueous sodium chloride solution and concentrated in vacuo.The residue was purified by chromatography on a silica gel column (usingdichloromethane:methanol=100:10 as the eluant) to afford the titlecompound (2.42 g, 4.13 mmol, 81%) as colorless amorphous solid.

¹H-NMR (270 MHz, DMSO-d₆): 11.36 (1H, s), 7.68 (1H, s), 6.90-7.44 (13H,m), 5.89 (1H, s), 5.55 (1H, d), 4.09 (1H, m), 4.04 (1H, d), 3.82 (2H,m), 3.74 (6H, s), 3.19 (2H, m), 1.99 (1H, m), 1.36 (1H, m), 1.17 (3H,s).

FAB-MAS (mNBA): 587 (M+H)⁺

Example 95′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-5-methyluridine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite

(Exemplification Compound Number 2-234)

A solution of the compound obtained in Example 8 (4.72 g, 8.05 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved under nitrogen atmosphere in anhydrousdichloromethane (142 ml). N,N-diisopropylamine (2.80 ml, 16.1 mmol) wasadded to the solution and then 2-cyanoethylN,N-diisopropylchlorophosphoramidite (2.16 ml, 9.66 mmol) was addeddropwise in an ice bath. The mixture was stirred at room temperature for6 hours. The reaction mixture was washed with saturated aqueous sodiumhydrogencarbonate solution and saturated aqueous sodium chloridesolution and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (using hexane:ethylacetate:triethylamine=50:50:1−hexane:ethyl acetate:triethylamine=30:60:1as the eluant) to afford the title compound (5.64 g, 7.17 mmol, 89%) asa colorless amorphous solid.

¹H-NMR (400 MHz, CDCl₃): 1.1-1.2 (15H, m), 1.4 (1H, m), 2.08 (1H, m),2.4 (2H, m), 3.2-4.0 (14H, m), 4.38 (2H, m), 4.47 (1H, m), 6.06 (1H, s),6.8-6.9 (4H, m), 7.2-7.5 (9H, m), 7.91 (1H, m).

FAB-MAS (mNBA): 787 (M+H)⁺

Example 10 3′,5′-Di-O-benzyl-2′-O,4′-C-ethylene-6-N-benzoyladenosine

(Exemplification Compound Number 1-23)

An aqueous 2N sodium hydroxide solution (5 ml) and mixture solution (5ml), said mixture solution comprised of pyridine:methanol:water=65:30:5,were added to compound obtained in Reference example 12 (238 mg, 0.30mmol) in pyridine:methanol:water=65:30:5 (5 ml) at 0° C. and the mixturewas stirred at room temperature for 15 minutes. The reaction mixture wasneutralized with 1N hydrochloric acid and extracted with ethyl acetate(about 30 ml). The organic layer was washed with saturated aqueoussodium hydrogencarbonate solution (about 30 ml) and saturated aqueoussodium chloride solution (about 30 ml), dried over anhydrous magnesiumsulfate and then concentrated in vacuo. The residue was purified bychromatography on a silica gel column (usingdichloromethane:methanol=50:1 as the eluant) to afford a colorlessamorphous solid (133 mg, 0.23 mmol, 78%).

¹H-NMR (400 MHz, CDCl₃): 1.44 (1H, d, 13 Hz), 2.31 (1H, dd, 13 and 19Hz), 3.56 (1H, d, 11 Hz), 3.70 (1H, d, 11 Hz), 4.10 (2H, m), 4.24 (1H,s), 4.45 (1H, d, 12 Hz), 4.53-4.67 (4H, m), 6.52 (1H, s), 7.3 (10H, m),7.53 (2H, m), 7.62 (1H, m), 8.03 (2H, d, 7.6 Hz), 8.66 (1H, s), 8.78(1H, s), 9.00 (1H, brs).

FAB-MAS (mNBA): 578 (M+H)⁺

Example 11 2′-O,4′-C-Ethylene-6-N-benzoyladenosine

(Exemplification Compound Number 1-178)

A 1M boron trichloride solution (1.5 ml, 1.5 mmol) in dichloromethanewas slowly added dropwise to a solution of the compound obtained inExample 10 (116 mg, 0.20 mmol) in anhydrous methylenechloride (5 ml) at−78° C. and the mixture was stirred at −78° C. for 3 hours. To thereaction mixture was added a 1M boron trichloride solution (1.5 ml, 1.5mmol) in dichloromethane and the mixture was stirred for 2 hours. Themixture was slowly warmed to room temperature and then quickly cooled to−78° C. and then methanol (5 ml) was added to the mixture. The reactionmixture was slowly warmed to room temperature and concentrated in vacuo.The residue was purified by chromatography on a silica gel column (usingdichloromethane:methanol=9:1 as the eluant) to afford a white powder (49mg, 0.17 mmol, 84%).

¹H-NMR (400 MHz, CD₃OD): 1.45 (1H, dd, 4.3 and 13 Hz), 2.12 (1H, m),3.72 (1H, d, 12 Hz), 3.79 (1H, d, 12 Hz), 4.04 (1H, dd, 7.3 and 12 Hz),4.15 (1H, dt, 4.3 and 9.4 Hz), 4.36 (1H, d, 3.2 Hz), 4.43 (1H, d, 3.2Hz), 6.57 (1H, s), 7.57 (2H, m), 7.66 (1H, m), 8.09 (2H, d, 8.0 Hz),8.72 (1H, s), 8.85 (1H, s).

FAB-MAS (mNBA): 398 (M+H)⁺

Example 12 2′-O,4′-C-Ethyleneadenosine

(Exemplification Compound Number 1-7)

A solution of the compound obtained in Example 11 (14 mg, 0.035 mmol) inmethanol saturated with ammonia (1 ml) was allowed to stand overnight.The mixture was concentrated and the residue was purified bychromatography on a silica gel column (usingdichloromethane:methanol=10:1 as the eluant) to afford a white powder(10 mg, 0.034 mmol, 98%).

¹H-NMR (400 MHz, CD₃OD): 1.32 (1H, dd, 4 and 13 Hz), 2.04 (1H, dt, 7.4and 12 Hz), 3.53 (1H, dd, 5 and 12 Hz), 3.61 (1H, dd, 5.2 and 12 Hz),3.90 (1H, dd, 7.4 and 12 Hz), 3.97 (1H, dt, 4 and 12 Hz), 4.15 (1H, d,3.1 Hz), 4.21 (1H, d, 3.1 Hz), 5.27 (1H, t, 5.2 Hz), 5.39 (1H, d, 3.1Hz), 6.33 (1H, s), 7.29 (2H, s), 7.66 (1H, m), 8.14 (1H, s), 8.42 (1H,s).

FAB-MAS (mNBA): 294 (M+H)⁺

UV (λmax): 260 (pH7), 260 (pH1), 258 (pH13)

Example 13 5′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-6-N-benzoyladenosine

(Exemplification Compound Number 1-31)

A solution of the compound obtained in Example 11 (14 mg, 0.035 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved in anhydrous pyridine (1 ml) under nitrogenatmosphere and 4,4′-dimethoxytritylchloride (18 mg, 0.053 mmol) wasadded to the solution and the mixture was stirred at 40° C. for 5 hours.A small amount of methanol was added to the reaction mixture and thenthe solvent was evaporated in vacuo. The residue was partitioned betweenwater and chloroform and the organic layer was washed with saturatedaqueous sodium hydrogencarbonate solution and saturated aqueous sodiumchloride solution and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (usingdichloromethane:methanol=100:5 as the eluant) to afford the titlecompound (18 mg, 0.026 mmol, 73%) as a colorless amorphous solid.

¹H-NMR (400 MHz, CDCl₃): 1.63 (1H, m), 2.14 (1H, 7.5, 12, and 13 Hz),3.37 (1H, d, 11 Hz), 3.41 (1H, d, 11 Hz), 3.79 (6H, s), 4.10 (2H, m),4.48 (1H, d, 3.3 Hz), 4.59 (1H, d, 3.3 Hz), 6.54 (1H, s), 6.85 (4H, m),7.2-7.6 (12H, m), 8.02 (2H, m), 8.45 (1H, s), 8.82 (1H, s), 9.02 (1H,brs). FAB-MAS (mNBA): 700 (M+H)⁺

Example 145′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-6-N-benzoyladenosine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite

(Exemplification Compound Number 1-186)

A solution of the compound obtained in Example 13 (16 mg, 0.023 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved under nitrogen atmosphere in anhydrousdichloromethane (0.5 ml). Tetrazole N,N-diisopropylamine salt (10 mg)was added to the solution and then 2-cyanoethylN,N,N′,N′-tetraisopropylphosphoramidite (about 20 μl) was added dropwisein an ice bath. The mixture was stirred at room temperature overnight.The reaction mixture was washed with saturated aqueous sodiumhydrogencarbonate solution and saturated aqueous sodium chloridesolution and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (using dichloromethane:ethylacetate=2:1 as the eluant) to afford the title compound (20 mg, 0.022mmol, 97%) as a white solid.

¹H-NMR (400 MHz, CDCl₃): 1.0-1.2 (12H, m), 1.54 (1H, m), 2.15 (1H, m),2.33 (2H, m), 3.3-3.6 (6H, m), 3.80 (6H, s), 4.08 (2H, m), 4.65 (1H, m),4.75 (1H, m), 6.53 (1H, s), 6.84 (4H, m), 7.2-7.6 (12H, m), 8.01 (2H,m), 8.53 (1H, s), 8.83 (1H, s), 9.01 (1H, brs).

FAB-MAS (mNBA): 900 (M+H)⁺

Example 15 3′,5′-Di-O-benzyl-2′-O,4′-C-ethyleneuridine

(Exemplification Compound Number 2-10)

An aqueous 1N sodium hydroxide solution (2 ml) was added to a solutionof the compound obtained in Reference example 13 (194 mg, 0.292 mmol) inpyridine (3 ml) at 0° C. and the mixture was stirred at room temperaturefor 30 minutes. The reaction mixture was neutralized with 1Nhydrochloric acid and extracted with ethyl acetate (10 ml). The organiclayer was washed with saturated aqueous sodium hydrogencarbonatesolution and saturated aqueous sodium chloride solution, dried overanhydrous magnesium sulfate and then concentrated in vacuo. The residuewas purified by chromatography on a silica gel column (usingdichloromethane:methanol=100:3 as the eluant) to afford an colorless oil(105 mg, 0.233 mmol, 80%).

¹H-NMR (400 MHz, CDCl₃): 1.36 (1H, m), 2.29 (1H, m), 3.63 (1H, d, 11Hz), 3.74 (1H, d, 11 Hz), 3.87 (1H, d, 2.9 Hz), 4.03 (2H, m), 4.29 (1H,d, 2.9 Hz), 4.49 (1H, d, 12 Hz), 4.50 (1H, d, 11 Hz), 4.53 (1H, d, 11Hz), 4.73 (1H, d, 12 Hz), 5.20 (1H, dd, 2 and 8 Hz), 6.04 (1H, s),7.2-7.4 (10H, m), 8.13 (1H, d, 8.2 Hz), 8.57 (1H, brs).

FAB-MAS (mNBA): 451 (M+H)⁺

Example 16 2′-O,4′-C-Ethyleneuridine

(Exemplification Compound Number 2-1)

A solution of the compound obtained in Example 15 (100 mg, 0.222 mmol)in methanol (4 ml) was stirred under hydrogen atmosphere at atmosphericpressure in the presence of 90 mg of 20% Pd(OH)₂ on carbon as ahydrogenation catalyst for 5 hours. The reaction mixture was filtered inorder to remove catalyst and the filtrate was concentrated in vacuo. Theresidue was purified by chromatography on a silica gel column (usingdichloromethane:methanol=10:1 as the eluant) to afford a colorless oil(45 mg, 0.167 mmol, 75%).

¹H-NMR (400 MHz, CD₃OD): 1.35 (1H, dd, 4 and 13 Hz), 2.13 (1H, ddd, 7,11 and 13 Hz), 3.66 (1H, d, 12 Hz), 3.73 (1H, d, 12 Hz), 3.91-4.08 (2H,m), 4.01 (1H, d, 3.2 Hz), 4.12 (1H, d, 3.2 Hz), 5.66 (1H, d, 8.2 Hz),6.00 (1H, s), 8.37 (1H, d, 8.2 Hz).

FAB-MAS (mNBA): 271 (M+H)⁺

Example 17 5′-O-Dimethoxytrityl-2′-O,4′-C-ethyleneuridine

(Exemplification Compound Number 2-15)

A solution of the compound obtained in Example 16 (28 mg, 0.104 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved in anhydrous pyridine (3 ml) under nitrogenatmosphere and 4,4′-dimethoxytritylchloride (50 mg, 0.15 mmol) was addedto the solution and the mixture was stirred at room temperatureovernight. A small amount of methanol was added to the reaction mixtureand then the solvent was evaporated in vacuo. The residue waspartitioned between water and chloroform and the organic layer waswashed with saturated aqueous sodium hydrogencarbonate solution andsaturated aqueous sodium chloride solution and concentrated in vacuo.The residue was purified by chromatography on a silica gel column (usingdichloromethane:methanol=100:3 as the eluant) to afford the titlecompound (25 mg, 0.044 mmol, 42%) as a colorless oil.

¹H-NMR (400 MHz, CD₃OD): 1.35 (1H, dd, 3 and 14 Hz), 2.03 (1H, ddd, 8,11 and 14 Hz), 2.46 (1H, d, 8 Hz), 3.36 (1H, d, 11 Hz), 3.41 (1H, d, 11Hz), 3.80 (3H, s), 3.81 (3H, s), 3.97 (2H, m), 4.21 (1), 4.33 (1H, brm),5.31 (1H, m), 6.10 (1H, s), 6.86 (4H, m), 7.2-7.5 (9H, m), 8.27 (1H, d,8.2 Hz), 8.43 (1H, brs).

FAB-MAS (mNBA): 573 (M+H)⁺

Example 185′-O-Dimethoxytrityl-2′-O,4′-C-ethyleneuridine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite

(Exemplification Compound Number 2-233)

A solution of the compound obtained in Example 17 (6 mg, 0.0105 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved under nitrogen atmosphere in anhydrousdichloromethane (0.5 ml). Tetrazole N,N-diisopropylamine salt (3 mg) wasadded to the solution and then 2-cyanoethylN,N,N′,N′-tetraisopropylphosphoramidite (about 5 μl) was added dropwisein an ice bath. The mixture was stirred at room temperature overnight.The reaction mixture was washed with saturated aqueous sodiumhydrogencarbonate solution and saturated aqueous sodium chloridesolution and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (using dichloromethane:ethylacetate=2:1 as the eluant) to afford the title compound (8 mg) as awhite solid.

¹H-NMR (400 MHz, CDCl₃): 1.1-1.2 (13H, m), 2.09 (1H, m), 2.4 (2H, m),3.3-3.6 (6H, m) 3.81 (6H, m), 3.94 (2H, m), 4.35 (1H, m), 4.47 (1H, m),5.18 (1H, d, 8.2 Hz), 6.08 (1H, s), 6.86 (4H, m), 7.2-7.4 (9H, m), 8.31(1H, d, 8.2 Hz).

FAB-MAS (mNBA): 773 (M+H)⁺

Example 193′,5′-Di-O-benzyl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine

(Exemplification Compound Number 2-46)

An aqueous 1N sodium hydroxide solution (5 ml) was added to a solutionof the compound obtained in Reference example 14 (310 mg, 0.396 mmol) inpyridine (5 ml) at 0° C. and the mixture was stirred at room temperaturefor 20 minutes. The reaction mixture was neutralized by dropwiseaddition of aqueous 20% acetic acid and extracted with dichloromethane.The dichloromethane layer was washed with saturated aqueous sodiumchloride solution and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (using dichloromethane:methanol=100:2 as the eluant) to afford the title compound (190 mg,0.334 mmol, 84%).

¹H-NMR (400 MHz, CDCl₃): 1.37 (1H, m), 1.58 (3H, s), 2.30 (1H, dt, 10and 13 Hz), 3.64 (1H, d, 11 Hz), 3.79 (1H, d, 11 Hz), 3.95 (1H, d, 3.0Hz), 4.04 (2H, dd, 2.3 and 10 Hz), 4.37 (1H, d, 3.0 Hz), 4.50 (1H, d, 12Hz), 4.56 (1H, d, 11 Hz), 4.61 (1H, d, 11 Hz), 4.76 (1H, d, 12 Hz), 6.11(1H, s), 7.2-7.5 (13H, m), 8.09 (1H, s), 8.29 (2H, m).

FAB-MAS (mNBA): 568 (M+H)⁺

Example 20 2′-O,4′-C-Ethylene-4-N-benzoyl-5-methylcytidine

(Exemplification Compound Number 2-226)

A 1M boron trichloride solution (1.6 ml) in dichloromethane was addeddropwise to a solution of the compound obtained in Example 19 (120 mg,0.211 mmol) in anhydrous dichloromethane (5 ml) at −78° C. and themixture was stirred at −78° C. for 4 hours. Methanol (1 ml) was slowlyadded dropwise to the mixture and the mixture was stirred for 10minutes. The pH of the reaction mixture was adjusted to 7-8 by dropwiseaddition of saturated aqueous sodium hydrogencarbonate solution. Thereaction mixture was warmed to room temperature and concentrated invacuo. The residue was purified by chromatography on a silica gel column(using dichloromethane:methanol=100:6 as the eluant) to afford the titlecompound (29 mg, 0.075 mmol, 36%) as a white solid.

¹H-NMR (400 MHz, d-DMSO): 1.24 (1H, m), 2.01 (3H, s), 2.0 (1H, m), 3.54(1H, dd, 5.4 and 12 Hz), 3.64 (1H, dd, 5.4 and 12 Hz), 3.88 (3H, m),4.10 (1H, m), 5.36 (1H, d, 5.4 Hz), 5.49 (1H, t, 5.0 Hz), 5.95 (1H, s),7.4-7.6 (3H, m), 8.21 (2H, m), 8.49 (1H, s), 13.17 (1H, brs).

FAB-MAS (mNBA): 388 (M+H)⁺

Example 215′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine

(Exemplification Compound Number 2-51)

A solution of the compound obtained in Example 20 (44 mg, 0.114 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved in anhydrous pyridine (1 ml) under nitrogenatmosphere and 4,4′-dimethoxytritylchloride (60 mg, 0.177 mmol) wasadded to the solution and the mixture was stirred at room temperatureovernight. A small amount of methanol was added to the reaction mixtureand then the solvent was evaporated in vacuo. The residue waspartitioned between water and chloroform. The organic layer was washedwith saturated aqueous sodium hydrogencarbonate solution and saturatedaqueous sodium chloride solution and concentrated in vacuo. The residuewas purified by chromatography on a silica gel column (usingdichloromethane:methanol=100:4 as the eluant) to afford the titlecompound (73 mg, 0.106 mmol, 93%) as a colorless oil.

¹H-NMR (400 MHz, CDCl₃): 1.46 (1H, m), 1.49 (3H, s), 2.06 (1H, m), 2.59(1H, d, 8.6 Hz), 3.36 (1H, d, 11 Hz), 3.39 (1H, d, 11 Hz), 3.80 (3H, s),3.81 (3H, s), 3.99 (2H, m), 4.30 (1H, d, 3.3 Hz), 4.39 (1H, m), 6.12(1H, s), 6.85 (4H, m), 7.2-7.5 (12H, m), 8.03 (1H, s), 8.28 (2H, m).

FAB-MAS (mNBA): 573 (M+H)⁺

Example 225′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite

(Exemplification Compound Number 2-236)

A solution of the compound obtained in Example 21 (35 mg, 0.0507 mmol)in anhydrous pyridine was azeotropically refluxed in order to removewater. The product was dissolved under nitrogen atmosphere in anhydrousdichloromethane (1 ml). Tetrazole N,N-diisopropylamine salt (17 mg) wasadded to the solution and then 2-cyanoethylN,N,N′,N′-tetraisopropylphosphoramidite (32 μA, 0.1 mmol) was addeddropwise in an ice bath. The mixture was stirred at room temperatureovernight. The reaction mixture was washed with saturated aqueous sodiumhydrogencarbonate solution and saturated aqueous sodium chloridesolution and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (using dichloromethane:ethylacetate=2:1 as the eluant) to afford the title compound (40 mg, 0.0445mmol, 89%) as a white solid.

¹H-NMR (400 MHz, CDCl₃): 1.1-1.2 (12H, m), 1.36 (3H, s), 1.37 (1H, m),2.10 (1H, m), 2.36 (2H, m), 3.3-3.6 (6H, m), 3.81 (6H, m), 3.98 (2H, m),4.42 (1H, m), 4.49 (1H, m), 6.11 (1H, s), 6.88 (4H, m), 7.2-7.5 (12H,m), 8.14 (1H, s), 8.28 (2H, m).

FAB-MAS (mNBA): 890 (M+H)⁺

Example 23 2′-O,4′-C-Ethylene-5-methylcytidine

(Exemplification Compound Number 2-226)

A solution of the compound obtained in Example 20 (11.6 mg, 0.030 mmol)in methanol saturated with ammonia (2 ml) was allowed to standovernight. The mixture was concentrated to afford a white solid (8.5 mg,0.030 mmol).

¹H-NMR (400 MHz, d-DMSO): 1.20 (1H, m), 1.82 (3H, s), 1.97 (1H, m), 3.49(1H, dd, 5 and 12 Hz), 3.58 (1H, dd, 5 and 12 Hz), 3.85 (2H, m), 5.23(1H, d, 5 Hz), 5.32 (1H, t, 5 Hz), 5.84 (1H, s), 6.7 (1H, brs), 7.2 (1H,brs), 8.08 (1H, s).

FAB-MAS (mNBA): 284 (M+H)⁺

UV (λmax): 279 (pH7), 289 (pH1), 279 (pH13)

Example 24 3′,5′-Di-O-benzyl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine

(Exemplification Compound Number 1-24)

An aqueous 1N sodium hydroxide solution (2 ml) was added to a solutionof the compound obtained in Reference example 15 (about 200 mg) inpyridine (2 ml) and the mixture was stirred at room temperature for 15minutes. The reaction mixture was neutralized with 1N hydrochloric acidand extracted with ethyl acetate. The organic layer was washed withsaturated aqueous sodium hydrogencarbonate solution and saturatedaqueous sodium chloride solution, dried over anhydrous magnesium sulfateand then concentrated in vacuo. The residue was purified bychromatography on a silica gel column (usingdichloromethane:methanol=50:1 as the eluant) to afford a colorlessamorphous solid (20 mg, 0.036 mmol, 6%, 2 steps).

¹H-NMR (400 MHz, CDCl₃): 1.27 (3H, s), 1.29 (3H, s), 1.43 (1H, dd, 3 and13 Hz), 2.28 (1H, m), 2.59 (1H, qui, 6.9 Hz), 3.54 (1H, d, 11 Hz), 3.68(1H, d, 11 Hz), 4.03 (2H, m), 4.15 (1H, d, 3.0 Hz), 4.31 (1H, d, 3.0Hz), 4.45 (1H, d, 12), 4.56 (1H, d, 12 Hz), 4.61 (1H, d, 12 Hz), 4.63(1H, d, 12 Hz), 6.18 (1H, s), 7.2-7.4 (10H, m), 8.19 (1H, s), 11.93 (1H,brs).

FAB-MAS (mNBA): 560 (M+H)⁺

Example 25 2′-O,4′-C-Ethylene-2-N-isobutyryl guano sine

(Exemplification Compound Number 1-177)

A solution of the compound obtained in Example 24 (10 mg, 0.018 mmol) inmethanol (2 ml) was stirred under hydrogen atmosphere at atmosphericpressure in the presence of 20 mg of 20% Pd(OH)₂ on carbon as ahydrogenation catalyst for 5 hours. The reaction mixture was filtered inorder to remove catalyst and the filtrate was concentrated in vacuo. Theresidue was purified by chromatography on a silica gel column (usingdichloromethane:methanol=10:2 as the eluant) to afford a colorless oil(5 mg, 0.013 mmol, 72%).

¹H-NMR (400 MHz, CD₃OD): 1.21 (3H, s), 1.22 (3H, s), 1.41 (1H, dd, 4 and13 Hz), 2.18 (1H, m), 2.69 (1H, qui, 6.9 Hz), 3.69 (1H, d, 12 Hz), 3.76(1H, d, 12 Hz), 4.0 (2H, m), 4.26 (1H, d, 3.2 Hz), 4.30 (1H, d, 3.2 Hz),6.30 (1H, s), 8.40 (1H, s).

FAB-MAS (mNBA): 380 (M+H)⁺

Example 265′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine

(Exemplification Compound Number 1-35)

A solution of the compound obtained in Example 25 (5 mg, 0.013 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved in anhydrous pyridine (1 ml) under nitrogenatmosphere and to 4,4′-dimethoxytritylchloride (14 mg, 0.04 mmol) wasadded to the solution and the mixture was stirred at 40° C. for 3 hours.A small amount of methanol was added to the reaction mixture and thenthe solvent was evaporated in vacuo. The residue was purified bychromatography on a silica gel column (usingdichloromethane:methanol=100:6 as the eluant) to afford the titlecompound (4 mg, 0.0059 mmol, 45%) as colorless solid.

¹H-NMR (400 MHz, CDCl₃): 1.26 (3H, d, 1.4 Hz), 1.28 (3H, d, 1.4 Hz),1.66 (1H, m), 2.15 (1H, m), 2.59 (1H, qui, 6.9 Hz), 3.65 (1H, m), 3.78(1H, m), 4.06 (2H, m), 4.35 (1H, m), 4.38 (1H, d, 3.2 Hz), 6.23 (1H, s),6.8 (4H, m), 7.2-7.5 (9H, m), 8.01 (1H, s), 8.19 (1H, brs).

FAB-MAS (mNBA): 682 (M+H)⁺

Example 275′-O-Dimethoxytrityl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite

(Exemplification Compound Number 1-185)

A solution of the compound obtained in Example 26 (4 mg, 0.0058 mmol) inanhydrous pyridine was azeotropically refluxed in order to remove water.The product was dissolved under nitrogen atmosphere in anhydrousdichloromethane (0.5 ml). Tetrazole N,N-diisopropylamine salt (5 mg) wasadded to the solution and then 2-cyanoethylN,N,N′,N′-tetraisopropylphosphoramidite (9 μl, 0.03 mmol) was addeddropwise in an ice bath. The mixture was stirred at room temperature for1 hour. The reaction mixture was washed with saturated aqueous sodiumhydrogencarbonate solution and saturated aqueous sodium chloridesolution and concentrated in vacuo. The residue was purified bychromatography on a silica gel column (using dichloromethane:ethylacetate=2:1 as the eluant) to afford the title compound (4 mg) as awhite solid.

¹H-NMR (400 MHz, CDCl₃): 1.1-1.4 (19H, m), 2.1 (1H, m), 2.4 (2H, m), 2.6(1H, m), 3.3-3.6 (6H, m), 3.8 (6H, s), 4.0-4.6 (4H, m), 6.2 (1H, s), 6.8(4H, m), 7.2-7.5 (9H, m), 8.1 (1H, s).

Example 28 2′-O,4′-C-Ethyleneguanosine

(Exemplification Compound Number 1-5)

A solution of the compound obtained in Example 25 (0.5 mg) in methanolsaturated with ammonia (0.5 ml) was allowed to stand at 60° C. for 5hours. The mixture was concentrated to afford a white powder (0.4 mg).

FAB-MAS (mNBA): 310 (M+H)⁺UV (λmax): 255 (pH7), 256 (pH1), 258-266(pH13)

Example 29 Synthesis of Oligonucleotide Derivative

Synthesis of an oligonucleotide derivative was carried out using amechanical nucleic acid synthesiser (ABI model392 DNA/RNA synthesiser: aproduct of Perkin-Elmer Corporation) on a scale of 1.0 μmole. Thesolvents, reagents and concentrations of phosphoramidite in everysynthetic cycle are the same as those in the synthesis of naturaloligonucleotides. Solvents, reagents and phosphoramidites of the naturaltype nucleosides are products of PE Biosystems Corporation. Everymodified oligonucleotide derivative sequence was synthesized byrepetition of condensation of the compound obtained in Example 9 oramidites containing the 4 species of nucleic acid bases for nucleotidesynthesis with the 5′-hydroxy group of thymidine produced bydeprotection of the DMTr group of 5′-O-DMTr-thymidine (1.0 μmole) usingtrichloroacetic acid, wherein the 3′-hydroxy group of the thymidine wasattached to a CGP carrier. The synthetic cycle is as follows:

1) detritylation trichloroacetic acid/dichloromethane; 35 sec

-   2) coupling phosphoramidite (about 20 eq), tetrazole/acetonitrile;    25 sec or 10 min-   3) capping 1-methylimidazole/tetrahydrofuran, acetic    anhydride/pyridine/tetrahydrofuran; 15 sec-   4) oxidation iodine/water/pyridine/tetrahydrofuran; 15 sec

In the above cycle 2) when the compound obtained in Example 9 was usedthe reaction time was 10 minutes and when phosphoramidites were used thereaction time was 25 seconds.

After synthesis of a desired oligonucleotide derivative sequence, the5′-DMTr group was removed and then the carrier containing the desiredproduct was conventionally treated with concentrated aqueous ammoniasolution in order to detach the oligomer from the carrier and todeprotect the cyanoethyl group which is protecting the phosphate group.The amino protecting group in adenine, guanine and cytosine was removedfrom the oligomer. The oligonucleotide derivative was purified byreverse-phase HPLC (HPLC: LC-VP: a product of Shimazu Corp.; column:Wakopak WS-DNA: a product of Wako Pure Chemical Industry Ltd.) to affordthe desired oligonucleotide.

According to this synthetic method the following oligonucleotidesequence (which oligonucleotide is hereinafter referred to as“oligonucleotide 1”) was obtained (0.23 μmol, yield 23%).

5′-gcgttttttgct-3′ (exemplification of SEQ ID NO: 2 in the SEQUENCELISTING) wherein the sugar moiety of the thymidines at base numbers 4 to8 is 2′-O,4′-C ethylene.

Reference Example 13,5-Di-O-benzyl-4-trifluoromethanesulfonyloxymethyl-1,2-O-isopropylidene-α-D-erythropentofuranose

Anhydrous pyridine (0.60 ml, 7.5 mmol) was added was added to a solutionof3,5-di-O-benzyl-4-hydroxymethyl-1,2-O-isopropylidene-α-D-erythropentofuranose(2000 mg, 5.0 mmol) in anhydrous dichloromethane (50 ml) andtrifluoromethanesulfonic anhydride (1010 mg, 6.0 mmol) under nitrogenatmosphere at −78° C. and the mixture was stirred for 40 minutes. Thereaction mixture was partitioned between the methylenechloride andsaturated aqueous sodium hydrogencarbonate solution (about 100 ml). Theorganic layer was washed with saturated aqueous sodium hydrogencarbonatesolution (about 100 ml) and saturated aqueous sodium chloride solution(about 100 ml), dried over anhydrous magnesium sulfate and thenconcentrated in vacuo to give a white powder (2520 mg, 4.73 mmol, 95%)which was used in the next reaction without further purification.

¹H-NMR (400 MHz, CDCl₃): 1.34 (3H, s), 1.63 (3H, s), 3.48 (1H, d, 10Hz), 3.53 (1H, d, 10 Hz), 4.21 (1H, d, 5.0 Hz), 4.5 (4H, m), 4.74 (1H,d, 12 Hz), 4.80 (1H, d, 12 Hz), 5.01 (1H, d, 12 Hz), 5.73 (1H, d, 4.6Hz), 7.3 (10H, m).

Reference Example 23,5-Di-O-benzyl-4-cyanomethyl-1,2-O-isopropylidene-α-D-erythropentofuranose

The compound obtained in Reference example 1 (2520 mg, 4.73 mmol) wasdissolved in dimethylsulfoxide (50 ml) at 90° C. To the solution wasadded sodium cyanide (463 mg, 9.46 mmol) at room temperature and themixture was stirred at 50° C. for 3 hours. The reaction mixture waspartitioned between water (about 100 ml) and ethyl acetate (about 100ml). The organic layer was washed with saturated aqueous sodium chloridesolution (about 100 ml), dried over anhydrous magnesium sulfate and thenconcentrated in vacuo. The residue was purified by chromatography onsilica gel (using hexane:ethyl acetate=4:1) to give a colorless oil(1590 mg, 3.89 mmol, 82%).

¹H-NMR (400 MHz, CDCl₃): 1.34 (3H, s), 1.62 (3H, s), 2.88 (1H, d, 17Hz), 3.15 (1H, d, 17 Hz), 3.50 (1H, d, 10 Hz), 3.58 (1H, d, 10 Hz), 4.08(1H, d, 5.1 Hz), 4.52 (1H, d, 12 Hz), 4.56 (1H, d, 12 Hz), 4.57 (1H, m),4.58 (1H, d, 12 Hz), 4.76 (1H, d, 12 Hz), 5.73 (1H, d, 3.7 Hz), 7.3(10H, m).

Reference Example 33,5-Di-O-benzyl-4-formylmethyl-1,2-O-isopropylidene-α-D-erythropentofuranose

A 1.5M toluene solution of isobutylaluminum hydride (2 ml, 3.0 mmol) wasslowly added dropwise to a solution of the compound obtained inReference example 2 (610 mg, 1.49 mmol) in dichloromethane (10 ml) undernitrogen atmosphere at −78° C. and the mixture was stirred for 1 hour at−78° C. and then warmed to room temperature. To the reaction mixture wasadded methanol (5 ml) and saturated aqueous ammonium chloride solution(about 20 ml) and this mixture was stirred for 30 minutes. The reactionmixture was extracted with ethyl acetate (about 30 ml). The organiclayer was washed with saturated aqueous sodium hydrogencarbonatesolution (about 30 ml) and saturated aqueous sodium chloride solution(about 30 ml), dried over anhydrous magnesium sulfate and thenconcentrated in vacuo to give a product which was used in the nextreaction without further purification.

Reference Example 43,5-Di-O-benzyl-4-hydroxyethyl-1,2-O-isopropylidene-α-D-erythropentofuranose

NaBH₄ (7.6 mg, 0.2 mmol) was added to a solution of the compoundobtained in Reference example 3 (154 mg, 0.377 mmol) in ethanol (5 ml)and the mixture was stirred at room temperature for 1 hour. The reactionmixture was partitioned between ethyl acetate (about 10 ml) and water(about 10 ml) and the organic layer was washed with saturated aqueoussodium chloride solution (about 10 ml), dried over anhydrous magnesiumsulfate and then concentrated in vacuo. The residue was purified bychromatography on silica gel (using hexane:ethyl acetate=2:1) to give acolorless oil (117 mg, 0.284 mmol, 75%).

¹H-NMR (400 MHz, CDCl₃): 1.33 (3H, s), 1.66 (3H, s), 1.78 (1H, ddd, 4.0,8.5, 15 Hz), 2.51 (1H, ddd, 3.4, 6.4, 15 Hz), 3.31 (1H, d, 10 Hz), 3.54(1H, d, 10 Hz), 3.80 (2H, m), 4.13 (1H, d, 5.3 Hz), 4.43 (1H, d, 12 Hz),4.52 (1H, d, 12 Hz), 4.55 (1H, d, 12 Hz), 4.65 (1H, dd, 4.0, 5.3 Hz),4.77 (1H, d, 12 Hz), 5.77 (1H, d, 4.0 Hz), 7.3 (10H, m).

FABMS (mNBA): 415 (M+H)⁺, [α]_(D)+57.4° (0.91, methanol).

Reference Example 53,5-Di-O-benzyl-4-formyl-1,2-O-isopropylidene-α-D-erythropentofuranose

Oxalyl chloride (6.02 ml, 69.0 mmol) was added to methylenechloride (200ml) cooled at −78° C. A solution of dimethylsulfoxide (7.87 ml, 110mmol) in anhydrous methylenechloride (100 ml) was added dropwise to thissolution. After stirring for 20 minutes a solution of3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-erythropentofuranose (9210 mg,23.02 mmol) in anhydrous dichloromethane (100 ml) was added dropwise tothis mixture and the mixture was stirred for 30 minutes. Triethylamine(28 ml, 200 mmol) was added to this reaction mixture and the mixture wasslowly warmed to room temperature. The reaction mixture was partitionedbetween the dichloromethane and water (about 300 ml). The organic layerwas washed with water (about 300 ml) and saturated aqueous sodiumchloride solution (about 300 ml), dried over anhydrous magnesium sulfateand then concentrated in vacuo. The residue was purified bychromatography on silica gel (using hexane:ethyl acetate=5:1) to give acolorless oil (8310 mg, 20.88 mmol, 91%).

¹H-NMR (400 MHz, CDCl₃): 1.35 (3H, s), 1.60 (3H, s), 3.61 (1H, d, 11Hz), 3.68 (1H, d, 11 Hz), 4.37 (1H, d, 4.4 Hz), 4.46 (1H, d, 12 Hz),4.52 (1H, d, 12 Hz), 4.59 (1H, d, 12 Hz), 4.59 (1H, dd, 3.4, 4.4 Hz),4.71 (1H, d, 12 Hz), 5.84 (1H, d, 3.4 Hz), 7.3 (10H, m), 9.91 (1H, s).FABMS (mNBA): 397 (M−H)⁺, 421 (M+Na)⁺, [α]_(D)+27.4° (0.51, methanol).

Reference Example 63,5-Di-O-benzyl-4-vinyl-1,2-O-isopropylidene-α-D-erythropentofuranose

A 0.5M toluene solution of Tebbe reagent (44 ml, 22 mmol) was added to asolution of the compound obtained in Reference example 5 (8310 mg, 20.88mmol) in anhydrous tetrahydrofuran (300 ml) under nitrogen atmosphere at0° C. and the mixture was stirred at 0° C. for 1 hour. Diethyl ether(300 ml) was added to the reaction mixture and then added 0.1N aqueoussodium hydroxide solution (20 ml) was slowly added. The mixture wasfiltrated through celite in order to remove precipitates and theprecipitates were washed with diethyl ether (about 100 ml). The organiclayer was dried over anhydrous magnesium sulfate and then concentratedin vacuo. The residue was purified by chromatography on basic aluminausing dichloromethane to afford crude product which was further purifiedby chromatography on silica gel (using hexane:ethyl acetate=8:1-5:1) togive a colorless oil (5600 mg, 14.14 mmol, 68%).

¹H-NMR (400 MHz, CDCl₃): 1.28 (31-1, s), 1.52 (3H, s), 3.31 (1H, d, 11Hz), 3.34 (1H, d, 11 Hz), 4.25 (1H, d, 4.9 Hz), 4.40 (1H, d, 12 Hz),4.52 (1H, d, 12 Hz), 4.57 (1H, dd, 3.9, 4.9 Hz), 4.59 (1H, d, 12 Hz),4.76 (1H, d, 12 Hz), 5.25 (1H, dd, 1.8, 11 Hz), 5.52 (1H, dd, 1.8, 18Hz), 5.76 (1H, d, 3.9 Hz), 6.20 (1H, dd, 11, 18 Hz), 7.3 (10H, m).

FABMS (mNBA): 419 (M+Na)⁺.

Reference Example 73,5-Di-O-benzyl-4-hydroxyethyl-1,2-O-isopropylidene-α-D-erythropentofuranose

A 0.5M tetrahydrofuran solution of 9-BBN (9-borabicyclo[3.3.1]nonane)(80 ml, 40 mmol) was added dropwise to a solution of the compoundobtained in Reference example 6 (5500 mg, 13.89 mmol) in anhydroustetrahydrofuran (200 ml) under nitrogen atmosphere and the mixture wasstirred at room temperature overnight. Water was added to the reactionmixture until evolution of gas ceased, 3N aqueous sodium hydroxidesolution (30 ml) was added and then slowly 30% aqueous hydrogen peroxidesolution was added keeping between 30 and 50° C. This mixture wasstirred for 30 minutes and partitioned between saturated aqueous sodiumchloride solution (about 200 ml) and ethyl acetate (200 ml). The organiclayer was washed with neutral phosphoric acid buffer solution (about 200ml) and saturated aqueous sodium chloride solution (about 200 ml) anddried over anhydrous magnesium sulfate and then concentrated in vacuo.The residue was purified by chromatography on silica gel (usinghexane:ethyl acetate=2:1-1:1) to give a colorless oil (5370 mg, 12.97mmol, 93%).

¹H-NMR (400 MHz, CDCl₃): 1.33 (3H, s), 1.66 (3H, s), 1.78 (1H, ddd, 4.0,8.5, 15 Hz), 2.51 (1H, ddd, 3.4, 6.4, 15 Hz), 3.31 (1H, d, 10 Hz), 3.54(1H, d, 10 Hz), 3.80 (2H, m), 4.13 (1H, d, 5.3 Hz), 4.43 (1H, d, 12 Hz),4.52 (1H, d, 12 Hz), 4.55 (1H, d, 12 Hz), 4.65 (1H, dd, 4.0, 5.3 Hz),4.77 (1H, d, 12 Hz), 5.77 (1H, d, 4.0 Hz), 7.3 (10H, m).

FABMS (mNBA): 415 (M+H)⁺, [α]_(D)+57.4° (0.91, methanol).

Reference Example 83,5-Di-O-benzyl-4-(p-toluenesulfonyloxyethyl)-1,2-O-isopropylidene-α-D-erythropentofuranose

Triethylamine (1.8 ml, 13 mmol), dimethylaminopyridine (30 mg, 0.25mmol), and p-toluenesulfonyl chloride (858 mg, 4.5 mmol) were added to asolution of the compound obtained in Reference example 4 which wasazeotropically refluxed with toluene (1035 mg, 2.5 mmol) in anhydrousdichloromethane (35 ml) under nitrogen atmosphere at 0° C. and themixture was stirred at room temperature overnight. The reaction mixturewas partitioned between the dichloromethane and saturated aqueous sodiumhydrogencarbonate solution (about 100 ml). The organic layer was washedwith saturated aqueous sodium hydrogencarbonate solution (about 100 ml)and saturated aqueous sodium chloride solution (about 100 ml) and driedover anhydrous magnesium sulfate and then concentrated in vacuo. Theresidue was purified by chromatography on silica gel (using hexane:ethylacetate=3:1) to give a colorless oil (1340 mg, 2.6 mmol, 94%).

¹H-NMR (400 MHz, CDCl₃): 1.33 (3H, s), 1.49 (3H, s), 1.99 (1H, dt, 7.6and 15 Hz), 2.47 (3H, s), 2.60 (1H, ddd, 5.7, 7.6, 15 Hz), 3.28 (1H, d,10 Hz), 3.45 (1H, d, 10 Hz), 4.11 (1H, d, 5.3 Hz), 4.32 (2H, m), 4.42(1H, d, 12 Hz), 4.50 (1H, d, 12 Hz), 4.54 (1H, d, 12 Hz), 4.62 (1H, dd,4.0, 5.2 Hz), 4.76 (1H, d, 12 Hz), 5.74 (1H, d, 4.0 Hz), 7.3 (12H, m),7.78 (2H, d, 8.3 Hz).

FAB-MAS (mNBA): 569 (M+H)⁺

Reference Example 91,2-Di-O-acetyl-3,5-di-O-benzyl-4-(p-toluenesulfonyloxyethyl)-α-D-erythropentofuranose

Acetic anhydride (1.88 ml, 20 mmol) and concentrated sulfuric acid (0.01ml) were added to a solution of the compound obtained in Referenceexample 8 (1340 mg, 2.36 mmol) in acetic acid (15 ml) and the mixturewas stirred at room temperature for 1 hour. The reaction mixture waspoured into water (60 ml) in an ice-bath and stirred for 30 minutes andthen partitioned between saturated aqueous sodium chloride solution(about 100 ml) and ethyl acetate (about 100 ml). The organic layer waswashed with neutral phosphoric acid buffer solution, saturated aqueoussodium hydrogencarbonate solution and saturated aqueous sodium chloridesolution and dried over anhydrous magnesium sulfate and thenconcentrated. The residue was purified by chromatography on silica gel(using hexane:ethyl acetate=2:1) to give a colorless oil (1290 mg, 2.11mmol, 89%, α:β=1:5).

¹H-NMR (400 MHz, CDCl₃): (β derivative) 1.86 (3H, s), 2.05 (3H, s), 2.08(1H, m), 2.18 (1H, m), 2.42 (3H, s), 3.30 (1H, d, 10 Hz), 3.33 (1H, d,10 Hz), 4.23 (1H, d, 5.1 Hz), 4.24 (2H, m), 4.42 (2H, s), 4.45 (1H, d,12 Hz), 4.55 (1H, d, 12 Hz), 5.28 (1H, d, 5.1 Hz), 6.01 (1H, s), 7.3(121-1, m), 7.73 (2H, d, 8.3 Hz).

FAB-MAS (mNBA): 613 (M+H)⁺

Reference Example 102′-O-Acetyl-3′,5′-di-O-benzyl-4′-p-toluenesulfonyloxyethyl-5-methyluridine

Trimethylsilylated thymine (500 mg, about 2 mmol), which was preparedaccording to a method of H. Vorbrggen, K. Krolikiewicz and B. Bennua(Chem. Ber., 114, 1234-1255 (1981)), was added to a solution of thecompound obtained in Reference example 9 (650 mg, 1.06 mmol) inanhydrous 1,2-dichloroethane (15 ml) at room temperature under nitrogenatmosphere. Trimethylsilyl trifluoromethanesulfonate (0.36 ml, 2 mmol)was added dropwise to the mixture and the mixture was stirred at 50° C.for 1 hour. Saturated aqueous sodium hydrogencarbonate solution (about50 ml) was added to the reaction mixture and the mixture was filteredthrough celite. Dichloromethane (about 50 ml) was added to the filtrate.The organic layer was washed with saturated aqueous sodiumhydrogencarbonate solution (about 50 ml) and saturated aqueous sodiumchloride solution (about 50 ml) and dried over anhydrous magnesiumsulfate and then concentrated in vacuo. The residue was purified bychromatography on silica gel (using hexane:ethyl acetate=1.2:1) to givea colorless amorphous solid (432 mg, 0.64 mmol, 60%).

¹H-NMR (400 MHz, CDCl₃): 1.52 (3H, d, 0.9 Hz), 1.94 (1H, dt, 7.5 and 15Hz), 2.06 (3H, s), 2.23 (1H, dt, 6.0 and 15 Hz), 2.42 (3H, s), 3.38 (1H,d, 10 Hz), 3.67 (1H, d, 10 Hz), 4.17 (2H, m), 4.36 (1H, d, 6.0 Hz), 4.41(1H, d, 12 Hz), 4.44 (1H, d, 12 Hz), 4.48 (1H, d, 12 Hz), 4.58 (1H, d,12 Hz), 5.39 (1H, dd, 5.1 and 6.0 Hz), 6.04 (1H, d, 5.1 Hz), 7.3 (12H,m), 7.73 (2H, dt, 1.8 and 8.3 Hz), 8.18 (1H, s).

FAB-MAS (mNBA): 679 (M+H)⁺

Reference Example 112′-O-Acetyl-3′,5′-di-O-benzyl-4′-p-toluenesulfonyloxyethyl-4-N-benzoylcytidine

Trimethylsilylated benzoylcytosine (300 mg, about 1.0 mmol), which wasprepared according to a method of H. Vorbrggen, K. Krolikiewicz and B.Bennua (Chem. Ber., 114, 1234-1255 (1981)), was added to a solution ofthe compound obtained in Reference example 9 (383 mg, 0.626 mmol) inanhydrous 1,2-dichloroethane (4 ml). Trimethylsilyltrifluoromethanesulfonate (0.18 ml, 0.995 mmol) at 0° C. was added tothe mixture and the mixture was stirred at 50° C. for 1 hour. Saturatedaqueous sodium hydrogencarbonate solution (about 10 ml) andmethylenechloride (about 20 ml) was added to the mixture and then themixture was stirred. The resulting white precipitates were filtered offthrough celite. The organic layer of the filtrate was washed withsaturated aqueous sodium chloride solution (about 20 ml) and dried overanhydrous magnesium sulfate and then concentrated in vacuo to give acolorless amorphous solid (397 mg, 83%).

¹H-NMR (400 MHz, CDCl₃): 8.70 (1H, br), 8.18 (1H, d, 7.4 Hz), 7.87 (2H,d, 7.5 Hz), 7.72 (2H, d, 8.3 Hz), 7.61-7.57 (1H, m), 7.51-7.48 (2H, m),7.43-7.21 (13H, m), 6.02 (1H, d, 2.9 Hz), 5.40 (1H, dd, 5.8, 2.9 Hz),4.57 (1H, d, 11 Hz), 4.39 (1H, d, 11 Hz), 4.32-4.28 (3H, m), 4.19-4.16(2H, m), 3.69 (1H, d, 11 Hz), 3.31 (1H, d, 11 Hz), 2.40 (3H, s),2.30-2.23 (1H, m), 2.06 (3H, s), 1.95-1.89 (1H, m)

FAB-MAS (mNBA): 768 (M+H)⁺

Reference Example 122′-O-Acetyl-3′,5′-di-O-benzyl-4′-p-toluenesulfonyloxyethyl-6-N-benzoyladenosine

Trimethylsilylated benzoyladenosine (500 mg, about 2.0 mmol), which wasprepared according to a method of H. Vorbrggen, K. Krolikiewicz and B.Bennua (Chem. Ber., 114, 1234-1255 (1981)), was added to a solution ofthe compound obtained in Reference example 9 (600 mg, 0.98 mmol) inanhydrous 1,2-dichloroethane (15 ml) at room temperature under nitrogenatmosphere. After dropwise addition of trimethylsilyltrifluoromethanesulfonate (0.36 ml, 2 mmol) to the mixture, the mixturewas stirred at 50° C. for 4 hour. Saturated aqueous sodiumhydrogencarbonate solution (about 50 ml) and dichloromethane (50 ml)were added to the reaction mixture and the mixture was partitionedbetween these two layers. The organic layer was washed with saturatedaqueous sodium hydrogencarbonate solution (about 50 ml) and saturatedaqueous sodium chloride solution (about 50 ml) and dried over anhydrousmagnesium sulfate and then concentrated in vacuo. The residue waspurified by chromatography on silica gel (usingdichloromethane:methanol=50:1) to give a colorless amorphous solid (405mg, 0.51 mmol, 52%).

¹H-NMR (400 MHz, CDCl₃): 2.0 (1H, m), 2.06 (3H, s), 2.32 (1H, dt, 6.0and 15 Hz), 2.40 (3H, s), 3.36 (1H, d, 10 Hz), 3.58 (1H, d, 10 Hz), 4.22(2H, m), 4.39 (1H, d, 12 Hz), 4.45 (1H, d, 12 Hz), 4.47 (1H, d, 12 Hz),4.59 (1H, d, 12 Hz), 4.62 (1H, d, 5.6 Hz), 5.94 (1H, dd, 4.5 and 5.6Hz), 6.21 (1H, d, 4.5 Hz), 7.2-7.3 (12H, m), 7.54 (2H, m), 7.62 (1H, dt,1.2 and 6.2 Hz), 7.72 (2H, d, 8.3 Hz), 8.02 (2H, m), 8.21 (1H, s), 8.75(1H, s), 8.97 (1H, brs).

FAB-MAS (mNBA): 792 (M+H)⁺

Reference Example 132′-O-Acetyl-3′,5′-di-O-benzyl-4′-p-toluenesulfonyloxyethyl-uridine

Trimethylsilylated uracil (200 mg, about 0.8 mmol), which was preparedaccording to a method of H. Vorbrggen, K. Krolikiewicz and B. Bennua(Chem. Ber., 114, 1234-1255 (1981)), was added to a solution of thecompound obtained in Reference example 9 (200 mg, 0.327 mmol) inanhydrous 1,2-dichloroethane (8 ml) at room temperature under nitrogenatmosphere. After dropwise addition of trimethylsilyltrifluoromethanesulfonate (0.145 ml, 0.8 mmol) to the mixture, themixture was stirred at 70° C. for 1 hour. Saturated aqueous sodiumhydrogencarbonate solution (about 10 ml) was added to the reactionmixture, the mixture was filtered through celite and dichloromethane(about 10 ml) was added to the filtrate. The organic layer was washedwith saturated aqueous sodium hydrogencarbonate solution and saturatedaqueous sodium chloride solution and dried over anhydrous magnesiumsulfate and then concentrated in vacuo. The residue was purified bychromatography on silica gel (using dichloromethane:methanol=100:2) togive a colorless oil (199 mg, 0.299 mmol, 92%).

¹H-NMR (400 MHz, CDCl₃): 1.94 (1H, dt, 7.4 and 15 Hz), 2.07 (3H, s),2.23 (1H, dt, 5.9 and 15 Hz), 2.43 (3H, s), 3.36 (1H, d, 10 Hz), 3.65(1H, d, 10 Hz), 4.17 (2H, dd, 6 and 7 Hz), 4.31 (1H, d, 5.9 Hz), 4.38(1H, d, 11 Hz), 4.39 (1H, d, 11 Hz), 4.40 (1H, d, 11 Hz), 4.58 (1H, d,11 Hz), 5.29 (1H, dd, 2.4 and 8.2 Hz), 5.33 (1H, dd, 4.5 and 6 Hz), 6.00(1H, d, 4.5 Hz), 7.2-7.4 (12H, m), 7.61 (1H, d, 8.2 Hz), 7.74 (1H, d,8.3 Hz), 8.14 (1H, brs).

FAB-MAS (mNBA): 665 (M+H)⁺

Reference Example 142′-O-Acetyl-3′,5′-di-O-benzyl-4′-p-toluenesulfonyloxyethyl-4-N-benzoyl-5-methylcytidine

Trimethylsilylated benzoyl 5-methylcytosine (400 mg, about 1.2 mmol),which was prepared according to a method of H. Vorbrggen, K.Krolikiewicz and B. Bennua (Chem. Ber., 114, 1234-1255 (1981)) was addedto a solution of the compound obtained in Reference example 9 (400 mg,0.653 mmol) in anhydrous 1,2-dichloroethane (6 ml). After addition oftrimethylsilyl trifluoromethanesulfonate (0.180 μl, 1.0 mmol) to themixture at 0° C., the mixture was stirred at 50° C. for 1 hour. Thereaction mixture was warmed to room temperature. Saturated aqueoussodium hydrogencarbonate solution (about 5 ml) and methylenechloride(about 10 ml) were) added to the reaction mixture and the mixture wasstirred. The mixture was filtered through celite in order to removewhite precipitates. The organic layer of the filtrate was washed withsaturated aqueous sodium chloride solution and dried over anhydrousmagnesium sulfate and then concentrated in vacuo to give a colorlessamorphous solid (320 mg, 0.409 mmol, 63%).

¹H-NMR (400 MHz, CDCl₃): 1.68 (3H, s), 1.95 (1H, dt, 7.3 and 15 Hz),2.07 (3H, s), 2.25 (1H, dt, 6 and 15 Hz), 2.43 (3H, s), 3.40 (1H, d, 10Hz), 3.71 (1H, d, 10 Hz), 4.18 (2H, m), 4.37 (1H, d, 5.8 Hz), 4.42 (1H,d, 12 Hz), 4.46 (1H, d, 12 Hz), 4.51 (1H, d, 12 Hz), 4.61 (1H, d, 12Hz), 5.42 (1H, dd, 4.9 and 5.8 Hz), 6.07 (1H, d, 4.9 Hz), 7.2-7.6 (17H,m), 7.74 (2H, d, 8.3 Hz), 8.28 (2H, d, 7.0 Hz).

FAB-MAS (mNBA): 782 (M+H)⁺

Reference Example 152′-O-Acetyl-3′,5′-di-O-benzyl-4′-p-toluenesulfonyloxyethyl-2-N-isobutyrylguanosine

Trimethylsilylated isobutyrylguanosine (650 mg, about 1.5 mmol), whichwas prepared according to a method of H. Vorbrggen, K. Krolikiewicz andB. Bennua (Chem. Ber., 114, 1234-1255 (1981)), was added to a solutionof the compound obtained in Reference example 9 (400 mg, 0.65 mmol) inanhydrous 1,2-dichloroethane (10 ml) at room temperature under nitrogenatmosphere. After addition of trimethylsilyl trifluoromethanesulfonate(0.2 ml, 1.2 mmol) to the mixture and the mixture was stirred at 50° C.for 4 hour. Saturated aqueous sodium hydrogencarbonate solution (about 5ml) was added to the reaction mixture and the organic layer was washedwith saturated aqueous sodium hydrogencarbonate solution and saturatedaqueous sodium chloride solution and dried over anhydrous magnesiumsulfate and then concentrated in vacuo to give a product which was usedin the next reaction without further purification.

Test Example 1 Tm Measurement Test

A sample solution (1000 μL) having a final concentration of NaCl of 100mM, sodium phosphate buffer solution (pH 7.2) of 10 mM, oligonucleotide(1) of 4 μM, and complementary DNA (hereinafter referred to asoligonucleotide (2)), having a sequence indicated by its complementarychain (sequence: 5′-agcaaaaaacgc-3′ (SEQ ID NO: 1 of the SEQUENCELISTING) or complementary RNA (hereinafter referred to asoligonucleotide (3)) having a sequence indicated by the sequence5′-agcaaaaaacgc-3′ (SEQ ID NO: 1 of the SEQUENCE LISTING), of 4 μM waswarmed in a boiling water bath and slowly cooled to room temperatureover the course of about two hours. The sample solution was then heatedand measured using a spectrophotometer (UV-3100PC: a product of ShimadzuCorp.). The sample was heated in a cell (cell thickness: 1.0 cm,cylindrical jacket type) by circulating water heated with an incubator(Haake FE2: a product of EKO Corp.), and the temperature was monitoredusing a digital thermometer (SATO SK1250MC). The temperature was raisedfrom 20° C. to 95° C. and the intensity of ultraviolet absorbance at themaximum absorption wavelength in the vicinity of 260 nm was measured foreach 1° C. increase in temperature. Naturally-occurring DNA (hereinafterreferred to as oligonucleotide (4)) having the sequence indicated by thesequence 5′-gcgttttttgct-3′ (Sequence No. 2 of the Sequence Listing),which is the same sequence as oligonucleotide (1) (compound of Example29), was used as the control, and the same procedure was performed.

The temperature at which the amount of change per 1° C. reached amaximum was taken to be Tm (melting temperature), and the complementarychain formation ability of the oligonucleotide analogue was evaluated atthis temperature.

The following shows the results of measuring the Tm values ofoligonucleotide (4) (naturally-occurring DNA) and oligonucleotide (1)(Compound of Example 29) relative to oligonucleotide (2) (complementaryDNA) and oligonucleotide (3) (complementary RNA).

TABLE 3 Tm (° C.) Compound Oligonucleotide (2) Oligonucleotide (3)Oligonucleotide (4) 48 44 Oligonucleotide (1) 61 75

As is clear from the above table, the oligonucleotide analogue of thepresent invention exhibited a remarkably higher Tm as well as remarkablyhigher complementary chain formation ability as compared withnaturally-occurring DNA.

Test Example 2 Measurement of Nuclease Enzyme Resistance

Exonuclease or endonuclease was mixed into a buffer solution ofoligonucleotide held at 37° C. for 15 minutes. The mixed solution wasthen held at 37° C. for a predetermined amount of time. Ethylenediaminetetraacetic acid (EDTA) was added to a portion of the mixed solution andthe mixture was heated at 100° C. for 2 minutes in order to stop thereaction. The amount of oligonucleotide remaining in the mixture wasdetermined by reverse phase high-performance liquid columnchromatography, and the time-based changes in the amount ofoligonucleotide in the presence of nuclease were measured.

The oligonucleotide analogues of the present invention demonstrateremarkable nuclease resistance.

Example 30 Synthesis of Oligonucleotide Derivative

According to the procedure of Example 29 the following oligonucleotidesequence (which oligonucleotide is hereinafter referred to as“oligonucleotide (5)”) was obtained.

5′-tcctctgtgcttggttctggcct-3′ (exemplification of SEQ ID NO: 3 in theSEQUENCE LISTING), wherein the sugar moiety of the thymidines andcytidines at base numbers 1 to 3 and 21 to 23 is 2′-O,4′-C ethylene.

Test Example 3 Inhibition of SNS/PN3 Gene Expression

Dorsal root ganglion (hereinafter “DRG”) was delivered from SD (SpragueDawley) rat of 16 fetal day age. DRG was incubated with PBS (sodiumphosphate buffer; 12 ml) containing 1 mg/ml collagenase for 30 min at37° C. After incubation, the supernatant of DRG was removed bycentrifugation. DRG was incubated with PBS (12 ml) containing 0.1%trypsin for 30 min at 37° C. After incubation DRG was dispersed byadding Dnase I solution (2 mg/ml in PBS, 60 μl) and pipetting in MEM(Minimum essential medium) containing 10% FCS (fetal calf serum), 28 mMglucose and 100 ng/ml NGF (resulting medium is hereinafter “FCS-MEM”).The resulting cell suspension (hereinafter “DRG cell”) was plated toeach well of 12 well plate in a ratio of 0.5 ml/well those well had beencoated with Poly-D-Lysin and filled with FCS-MEM (0.5 ml/well)containing 16 ng/ml laminin.

DRG cell was cultured in 5% CO₂ atmosphere at 37° C. On the dayfollowing the plating, half of medium (0.5 ml) was replaced with FCS-MEMcontaining 20 μM Ara-C.

On the 4th day following the plating, half of medium was replaced withFCS-MEM containing 40 μM oligonucleotide (5) (compound of Example 30).

After incubating 48 hours, RNA of the DRG cell was extracted. Using theRT-PCR (reverse transcript PCR) method, the quantity of the mRNA of theSNS/PN3 was measured (internal standard: TrkA (NGF receptor)). Theinhibition ability was evaluated by the following formula.

-   A=mRNA quantity of the compound treated well-   B=mRNA quantity of non treated well (control)    Inhibition rate(%)=(1−A/B)×100

TABLE 4 Compound inhibition rate (%) Oligonucleotide (5) 28As is clear from the above table, the oligonucleotide analogue of thepresent invention exhibited a remarkably high inhibition ability ofSNS/PN3 gene expression. The oligonucleotide analogue of the presentinvention is thus useful as a pain treatment drug.Industrial Applicability

The novel oligonucleotide analogue and nucleoside analogue of thepresent invention are useful as antisense or antigene pharmaceuticalshaving excellent stability, as detection agents (probes) of a specificgene, as primers for starting amplification or as intermediates fortheir production.

What is claimed is:
 1. An antigene oligonucleotide comprising two ormore nucleoside units, wherein at least one of said nucleoside units hasa structure of formula (2):

said nucleoside units being bonded through a phosphodiester bond or aphosphorothioate bond, wherein: A represents a methylene group; and B isa group selected from the group consisting of 6-benzoylaminopurin-9-yl,adeninyl, 2-isobutyrylamino-6-hydroxypurin-9-yl, guaninyl,2-oxo-4-benzoylamino-pyrimidin-1-yl, cytosinyl,2-oxo-5-methyl-4-benzoylamino-pyrimidin-1-yl, 5-methylcytosinyl,uracinyl and thyminyl groups.
 2. The antigene oligonucleotide accordingto claim 1, wherein the oligonucleotide analogue, B is an adeninylgroup.
 3. The antigene oligonucleotide according to claim 1, wherein theoligonucleotide analogue, B is a guaninyl group.
 4. The antigeneoligonucleotide according to claim 1, wherein the oligonucleotideanalogue, B is a uracinyl group.
 5. The antigene oligonucleotideaccording to claim 1, wherein the oligonucleotide analogue, B is acytosinyl group.
 6. The antigene oligonucleotide according to claim 1,wherein the oligonucleotide analogue, B is a 5-methylcytosinyl group. 7.A nucleoside analog represented by the following structural formula:

wherein B is adenine, guanine, thymine, 5-methyl-cytosine, cytosine, oruracil.
 8. The nucleoside analogue of claim 7, wherein the analogue isone of the following specific compounds: a)(1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(adenin-9-yl)-2,6-dioxabicyclo[3.2.1]octane,b)(1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(guanin-9-yl)-2,6-dioxabicyclo[3.2.1]octane,c)(1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(thymin-1-yl)-2,6-dioxabicyclo[3.2.1]octane,d)(1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(5-methyl-cytosin-1-yl)-2,6-dioxabicyclo[3.2.1]octane,e)(1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(cytosin-1-yl)-2,6-dioxabicyclo[3.2.1]octane,and f)(1R,5R,7R,8S)-8-hydroxy-5-(hydroxymethyl)-7-(uracil-1-yl)-2,6-dioxabicyclo[3.2.1]octane.9. A compound selected from the group consisting of2′-O,4′-C-ethyleneguanosine, 2′-O,4′-C-ethyleneadenosine,2′-O,4′-C-ethyleneuridine, 2′-O,4′-C-ethylene-5-methyluridine,2′-O,4′-C-ethylenecytidine, and 2′-O,4′-C-ethylene-5-methylcytidine. 10.A compound selected from the group consisting of2′-O,4′-C-ethyleneguanosine, 2′-O,4′-C-ethyleneadenosine,3′,5′-di-O-benzyl-2-O,4′-C-ethylene-6-N-benzoyladenosine,3′,5′-di-O-benzyl-2-O,4′-C-ethylene-2-N-isobutyrylguanosine,5′-O-dimethoxytrityl-2-O,4′-C-ethylene-6-N-benzoyladenosine,5′-O-dimethoxytrityl-2-O,4′-C-ethylene-2-N-isobutyrylguanosine,2′-O,4′-C-ethylene-2-N-isobutyrylguanosine,2′-O,4′-C-ethylene-6-N-benzoyladenosine,5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-6-N-benzoyladenosine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite,5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite, 2′-O,4′-C-ethyleneuridine,2′-O,4′-C-ethylene-5-methyluridine, 2′-O,4′-C-ethylenecytidine,2′-O,4′-C-ethylene-5-methylcytidine,3′,5′-di-O-benzyl-2′-O,4′-C-ethyleneuridine,5′-O-dimethoxytrityl-2′-O,4′-C-ethyleneuridine,3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-5-methyluridine,5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-5-methyluridine,3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-4-N-benzoylcytidine,5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoylcytidine,3′,5′-di-O-benzyl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine,5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine,2′-O,4′-C-ethylene-4-N-benzoylcytidine,2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine,5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-uridine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite,5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-5-methyluridine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite,5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoylcytidine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite, and5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine-3′-O-(2-cyanoethylN,N-diisopropyl)phosphoramidite.
 11. An oligonucleotide analoguecomprising two or more nucleoside units wherein at least one of saidnucleoside units is a structure of the formula:

wherein: B is selected from the group consisting of adenine, guanine,thymine, 5-methyl-cytosine, cytosine, and uracil.
 12. An oligonucleotideanalogue comprising two or more nucleoside units wherein at least one ofsaid nucleoside units is a structure of the formula:

wherein: A represents a methylene group; and B is selected from thegroup consisting 6-benzoylaminopurin-9-yl, adeninyl,2-isobutyrylamino-6-hydroxypurin-9-yl, guaninyl,2-oxo-4-benzoylamino-pyrimidin-1-yl, cytosinyl, 5-methylcytosinyl,uracinyl and thyminyl groups.
 13. An oligonucleotide analogue comprisingtwo or more nucleoside units wherein at least one of said nucleosideunits is a structure of the formula:

wherein: A represents a methylene group; and B is2-oxo-5-methyl-4-benzoylamino- pyrimidin-1-yl.